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Evaluation of bull performance based on in vitro fertilization studies Kurtu, Jamal A. 1997

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EVALUATION OF BULL PERFORMANCE BASED ON IN VITRO FERTILIZATION STUDIES By JAMAL A. KURTU B.Sc, University of Alexandria, Alexandria, Egypt, 1985 Food Science Diploma, B.C. Institute of Technology, Vancouver, Canada, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE We accept this thesis as conforming to the standard THE UNIVERSITY OF BRITISH COLUMBIA MAY 1997 ® Jamal M. A. Kurtu, 1997 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purpose may be granted by the head of my department or his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of AfffiAt Scr<ffl'C& The University of British Columbia Vancouver, Canada D a t e o^re/i f / ? / $ , / ? ? ? ABSTRACT The first part of this study investigated if differences among bulls as semen donors, and sperm concentrations affected cleavage rate of oocytes. Three levels of sperm concentration and their effect on cleaving oocytes were analyzed. Frozen-thawed semen samples from five Holstein bulls and oocytes (n=728) harvested from slaughterhouse ovaries were used in this experiment. Oocytes were matured in vitro with Hams' F-10 and estrus cow serum (ECS) media, then fertilized in vitro using Tyrode Albumin Lactate Pyruvate (TALP) media. The resulting embryos were subsequently cultured using Hams' F-10 and fetal calf serum (FCS) in the presence of oviductal cells . The three sperm concentrations used in the study were 2xl06, lxlO 6 and 0.5xl06 /ml. Approximately 56- 72 h after addition of sperm, cleavage as indicated by a 2-cell stage embryo was observed and recorded. A significant difference (P< 0.001) in cleavage rate was observed among the five bulls. Sperm concentration did not affect cleavage rate. Bull x sperm interaction was also not significant (P>0.05). The in vivo fertility based on 60 - 90 d non-return rate (NRR) to first insemination did not correlate with the in vitro cleavage rate (P>0.05, r = 0.51). In the second part of the study, the same bulls were tested for their ability to cause further embryonic development beyond cleavage to the blastocyst stage. The resulting embryos were frozen at either the morula or blastocyst stage to study the freeze- thaw survival rate. Unlike in the first part of the study, the oocytes were matured in vitro using tissue culture medium (TCM 199) supplemented with superovulated cow serum (SCS), fertilized in vitro using Brackett and Oliphant (BO) media. The embryos were cultured for 7-9 d using TCM 199 medium supplemented with insulin and SCS in the presence of cumulus cell monolayers. Embryos that reached the morula or blastocyst stage were frozen in 0.25 cc straws by ii controlled freezing and thawing procedure using 1.6 M propylene glycol as a cryoprotectant. No significant differences (P>0.05) were observed among all five bulls in their ability to cause further embryonic development. In addition, the post thaw survival rate of embryos produced by all five bulls following freeze-thaw did not differ significantly (P>0.05). The in vivo fertility based on 60 - 90 d non-return rates (NRR) to the first insemination did not correlate with the in vitro embryonic development and freeze-thaw survival rate (P>0.05). The number and quality of embryos produced using the combination of TCM 199 medium and defined BO media with serum was found to be superior to the combination of Hams' F10 and TALP media with serum. iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES vii LIST OF PLATES viii LIST OF TABLES ix GLOSSARY OF COMMON ABBREVIATIONS x ACKNOWLEDGMENTS xi DEDICATION xii CHAPTER 1 2 GENERAL INTRODUCTION 2 CHAPTER 2 . . . .7 LITERATURE REVIEW 7 2.1. IN-VITRO FERTILIZATION (IVF) 7 2.2. THE OVARIES AND THE OOCYTES 8 2.2.1 Oocyte Maturation Process In-Vivo 9 2.2.2 In-Vitro Maturation (TVM) 10 2.2.3 Harvesting Oocytes From Ovaries 12 2.3 BULL SPERM PREPARATION FOR IVF 13 2.3.1 Capacitation And Acrosome Reaction Of Spermatozoa 14 2.4 FERTILIZATION, CLEAVAGE AND EMBRYONIC DEVELOPMENT 16 2.5 FACTORS AFFECTING CULTURE OF SPERM AND OOCYTES 18 2.6 IN-VITRO CULTURE (IVC) 19 2.6.1 Co-Culture With Somatic Cells 19 2.7 MEDIA FOR IVF.... 20 2.7.1. Inorganic Ions, Organic Compounds And Sera For Culture Media 20 2.8 MEASUREMENT OF REPRODUCTIVE EFFICIENCY 21 2.8.1 Non return rate (NRR) 22 2.8.2 Calving rate 22 2.8.3 Calving interval 23 2.8.4 Services Per Conception 23 2.9 EVALUATION OF BULL FERTILITY IN VITRO 23 2.9.1 Consecutive Sperm Motility 24 2.9.2 Sperm Cell Morphology 24 2.9.3 Staining of Live and Dead Sperm 24 2.9.4 Other Tests 25 iv 2.9.5 The Relationship Between In Vitro And In Vivo Fertility Of Bulls 25 2.10 THE FREEZING AND THAWING OF MAMMALIAN EMBRYOS 26 2.10.1 Principles Of Cryopreservation 27 2.10.2 Use of Cryoprotectants 29 2.10.3 Controlled Freezing and Thawing 30 2.10.4 Media Supplementation 30 2.10.5 Loading Embryos into Straws And The Freezing Machine 30 CHAPTER 3 33 BULLS AND SPERM CONCENTRATION EFFECT 33 ON BOVINE OOCYTESCLEAVAGE RATE 33 3.1 ABSTRACT 33 3.2 INTRODUCTION 34 3.3 MATERIALS AND METHODS 36 3.3.1 In Vitro Oocyte Maturation: 36 3.3.1.1 Collection of ovaries 36 3.3.1.2 Oocyte aspiration 36 3.3.2 Oviductal cell preparation: 37 3.3.3 Semen Preparation 38 3.3.4 In vitro Fertilization: 38 3.3.5 Assessing Cleavage and in vitro culture 39 3.3.6 Non return rate result 41 3.3.7 Statistical Analysis 41 3.4 RESULTS 41 3.4.1 Effect of Bull on cleavage rate of oocytes 41 3.4.2 Effect of sperm concentration on cleavage rate of oocytes 42 3.4.3 Non Return Rate 43 3.5 DISCUSSION 44 3.6 CONCLUSION 50 CHAPTER 4 52 EFFECT OF BULL ON IN VITRO EMBRYO DEVELOPMENT AND 52 EMBRYO VIABILITY AFTER FREEZE-THAW 52 4.1 ABSTRACT 52 4.2 INTRODUCTION 53 4.3 MATERIALS AND METHODS 54 4.3.1 In Vitro Oocyte Maturation: 54 4.3.1.1 Collection of ovaries 54 4.3.1.2 Oocyte aspiration and maturation 55 4.3.2 Semen Preparation and in vitro fertilization 56 4.3.3 In vitro culture and Assessing Embryo Development 57 4.3.4 Embryo Freezing And Thawing 64 4.3.. 5 Non return rate result 65 V 4.3.6 Statistical Analysis 65 4.4 RESULTS 66 4.4.1 Effect of bull on embryo development in Vitro 67 4.4.2 Effect of bull on Freeze-thaw survival rate of embryos 67 4.5 DISCUSSION 68 4.6 CONCLUSION 72 CHAPTER 5 73 IN VITRO SYSTEM IN RELATION TO EMBRYO DEVELOPMENT 73 5.1 ABSTRACT 73 5.2 INTRODUCTION AND RESULTS 74 5.3 POSSIBLE VARIATION INVOLVED IN THE TWO SYSTEMS 76 5.3.1 In- vitro Maturation (IVM) 79 5.3.2 In- vitro Fertilization (IVF) 81 5.3.3 In- vitro Culture (IVC) 83 5.4 CONCLUSION 84 CHAPTER 6. 86 GENERAL DISCUSSION 86 6.1 FUTURE SCOPE OF IVF 92 6.2 IMPROVING IN- VITRO BOVINE EMBRYO PRODUCTION 93 REFERENCES 96 APPENDIX A. STEPS INVOLVED IN BOVINE IN VITRO FERTILIZATION USING TALP METHOD 110 APPENDIX B. STEPS INVOLVED IN BOVINE IN VITRO FERTILIZATION USING BO METHOD I l l APPENDIX C. STEPS INVOLVED IN PREPARATION OF TALPS MEDIA AND ITS COMPOSITION 112 APPENDIX D. IVF PROCEDURE FOR BO METHOD 114 APPENDIX E. COMPOSITION OF BO MEDIA USED IN IVF FOR EMBRYONIC DEVELOPMENT 118 APPENDIX F. STEPS INVOLVED IN CALCULATING SPERM CONCENTRATION119 E X A M P L E OF CALCULATING SPERM CONCENTRATION FOR IVF (FROZEN VS. FRESH) 121 APPENDIX G. PROTOCOL FOR FREEZE-THAW SURVIVAL RATE OF EMBRYO AND SOLUTION PREPARATION 123 vi LIST O F FIGURES F I G U R E 3.1. B U L L D I F F E R E N C E S O N O O C Y T E C L E A V A G E R A T E vii LIST OF PLATES PLATE 3.1 CLEAVAGE OF THE ZYGOTE TWO-DAUGHTER CELLS 40 PLATE 4.1. IMMATURE OOCYTE IMMEDIATELY AFTER ASPIRATION AND MATURE OOCYTE WITH EXPANDED CUMULUS CELLS 58 PLATE 4.2. EARLY EMBRYONIC DEVELOPMENT 59 PLATE 4.3. MORULA STAGE EMBRYO APPROXIMATELY 5 - 7 DAYS AFTER INSEMINATION 60 PLATE 4.4. VARIOUS STAGES OF BLASTOCYST 61 PLATE 4.5. HATCHING BLASTOCYST AND ZONA FREE HATCHED EMBRYO .. 62 PLATE 4.6. DIFFERENT STAGES OF CULTURED CUMULUS CELLS 63 viii LIST OF TABLES TABLE 3.1: EFFECT OF SPERM CONCENTRATION WITHIN BULL ON CLEAVAGE RATE 43 TABLE 3.2: EFFECT OF SPERM CONCENTRATION (POOLED DATA) ON CLEAVAGE RATE 43 TABLE 3.3. NON RETURN RATE RESULTS OBTAINED FROM A l CENTER 44 TABLE 3.4. NON RETURN RESULTS AND CLEAVAGE RATE 44 TABLE 4.1: RESULTS OF IN VITRO EMBRYO DEVELOPMENT FOR FIVE BULLS .. 67 TABLE 4.2: NON RETURN RATE AND EMBRYONIC DEVELOPMENT 67 TABLE 4.3: EFFECT OF BULL ON FREEZE-THAW SURVIVAL RATE OF EMBRYOS67 TABLE 5.1. RESULTS OF EMBRYONIC DEVELOPMENT FOR THE TALP METHOD 75 TABLE 5.2. RESULTS OF EMBRYONIC DEVELOPMENT FOR THE BO METHOD ... 75 ix GLOSSARY OF COMMON ABBREVIATIONS The explanatory vocabulary list in the text is given in alphabetical order: p.1 = Microliter A = Aromour AI = Artificial insemination AR = Acrosome reaction BO = Brackett and Oliphant BOEC = Bovine oviductal epithelial cells BSA = Bovine Serum Albumin cAMP = Cyclic adenosine monophospate CL = Corpus luteum d Day (days) ECS = Estrus cow serum EGF = Epidermal growth factor FCS = Fetal calf serum FSH = Follicle-stimulating hormone GAG Glycosaminoglycans GV = Germinal vesicle GVBD = Germinal vesicle break down h — Hours hCG = Human chorionic gonadotropin HEPES = Hydroxyethylpiperazine-ethanesulfonic acid HIS = High ionic strength solutions IVC = In Vitro culture IVF - - In-vitro fertilization IVM = In Vitro maturation LH = Luteinizing hormone mg = Milligram Mil = Metaphase II min = minute mm = Millimeter MPF = Maturation promoting factor NRR Non -return rates PG MEDIUM -- Pyruvate - Glucose Medium PG = Propylene glycol :l,2-propanediol (PROH) SCS Superovulated cow serum TALP - Tyrode Albumin Lactate Pyruvate TCM Tissue culture medium A C K N O W L E D G M E N T S I thank Dr. R. Rajamahendran for his full support and ideas to accomplish my thesis as well as in assisting me during my study. I also thank Divaker Ambrose for his countless assistance in helping me to edit the first part of this thesis and for his training in technical aspects of IVF. I am obliged to Mohan Mahesh for his time and effort to read and correct my thesis for essay comprehensibility. The technical contribution that has been given by K. Sivakumaran in the first part of my thesis and his teaching skill in IVF procedure is greatly appreciated. I truly admire Sylvia Leung, Patrick Charagu and Samuel Aggrey for their assistance in solving statistical analysis of data. I appreciate Lisa Stephans for her time and effort to read and edit my thesis. I am indebted to BCAI center , Langley, B.C., for providing frozen semen and bull fertility data. I am also indebted to J & L Beef , Surrey, B.C. for their generous supply of bovine ovaries. I am thankful to my advisory committee, especially to Dr. K. M. Cheng for his constructive feedback in editing and correcting my thesis. I am grateful to Dr. J.A. Shelford and Dr. Gregory Lee to be in my examiner committees. I am appreciative to faculty members, staffs, students of the Department of Animal Science, and the staffs at the Animal Science Teaching & Research facility at south campus for their friendly approaches. My especial thanks will be extended to Ted Cathcart, Cathleen Nichols, Chris Shingera, Klara Shekhtman and Ian Smith for their moral and emotional support. Truly speaking, they were my forefronts in teaching me patience in my work environment. My acknowledgment attenuates to Gills Galazy who gave me a basic knowledge in computer skill during my study. I thank him for all technical supports he offered me during the past years. Finally, I am beholden to my wife for her support, understanding and encouragement for what I am today. I thank her for taking care of our children Nouria and Tasneem. I also thank my two daughters for their indirect support by being nice to their mother when I was engrossed in my study. xi DEDICATION To My Wife, Fatima And My Children, Nouria And Tasneem For Their Affection And Benevolence For My Purpose In Life xii CHAPTER 1. GENERAL INTRODUCTION CHAPTER 1. GENERAL INTRODUCTION Since the birth of the first in vitro fertilized calf (Brackett et, al. 1982), in vitro fertilization (IVF) has been utilized for several purposes among which evaluation of bull fertility has gained reasonable attention. The IVF technique has been employed for the large scale production of cattle embryos, yet the efficiency of production is low (Gordon and Lu, 1990). In vitro embryo production is a complicated process that compromises oocyte recovery, fertilization, embryo storage and establishment of pregnancies. Some of the factors that influence the success rate of in-vitro embryo production in cattle are: composition of culture media, the sperm-oocyte selection, composition of hormonal supplements, environmental factors of the in vitro system, and the methods of embryo cryopreservation. Even though pregnancies and births were observed using these techniques in many laboratories, the quality of embryos produced in vitro are lower than those produced in vivo (Leibfried-Rutledge et al., 1989 ). This indicates that the techniques for in vitro embryo production still require considerable improvement before being fully applied in research and commercial production. The in-vitro embryo production system in general and the male-female gamete factors in particular must be standardized to overcome all drawbacks involved in this procedure (Brackett, 1992). Since many unknown events are involved in the fertilization process of egg both in-vivo and in-vitro, it would be ideal to study all these factors separately. As well as the factors influencing IVF, the effects of bull differences on embryo production and development are examined in this study. 2 There is a similarity in morphological and physiological events of in-vivo and in-vitro matured oocytes, but the process that occurs in-vitro is quicker than in vivo (King et al, 1986). On the other hand, the quality of in-vitro produced embryos is inferior compared to those produced in-vivo. In vitro produced embryos are morphologically darker, form an early blastocoeles, have fewer cells per embryo and are highly susceptible to freeze-thaw damage (Greve, etal, 1993). In terms of cryopreservation, the first successful freezing of mammalian embryos was done by Whittingham (1971). Freezing procedures developed during the past decade have enabled high freeze-thaw survival rates for non surgically collected bovine morulae and blastocysts (Niemann, 1991). It has been suggested that some chemical components of the spermatozoa are affected by cryopreservation (Buhr and Zhoa, 1995). Spermatozoa are highly sensitive to any variation that occurs during cryopreservation, particularly those from sub-fertile bulls which in turn might affect the success rate of embryo production in-vitro. It is assumed that the spermatozoa from highly fertile bulls would tolerate cryopreservation and produce healthier embryos. Therefore, it is hypothesized that embryos produced by highly fertile bulls would tolerate freeze-thaw and this could be used as a performance test for bulls. Successful pregnancies following artificial insemination (Al) are considered to be the most valid test for evaluating bull fertility in the field. Nevertheless, this methods is time consuming and expensive. To date, the standard procedures for determining bull fertility are based on 60-90 d. non return rates (NRR) and a standard laboratory semen evaluation. However, these methods are not accurate. Recent reports indicate that bull differences for early fertilization, oocyte cleavage and early embryonic development can be assessed using the IVF technique (Hillery et al., 1990, Eyestone and First, 1989a). It was suggested that a 3 valuable males can be selected using such IVF parameters (Kroetsh, et al., 1992, T. Greve, et al, 1993). It is necessary to test each bull to determine the number of sperm that are used for IVF (Leibfried-Ruteledge et al., 1989) since each bull has its own success rate in fertilization and embryonic development (Hillary et al. 1990; Shi et al, 1990). IVF and subsequent embryonic development are not only influenced by bull differences, but also affected by sperm concentration, sperm preparation and culture media (Shamsuddin and Larsson, 1993). A correlation has been established between a bull's ability to produce embryos in vitro and 60-90 d NRR based on field data of the same bull (Hillery et al., 1990, Shamsuddin and Larsson, 1993, Bousquet et al., 1983). Even though bull differences in IVF are well documented, the correlation between NRR and IVF results has not been convincing (Graule et ah, 1995). The IVF system could be an ideal method to evaluate bull performance in addition to the methods discussed earlier. This can be accomplished in a cost effective manner if slaughterhouse ovaries can be used as starting material. Therefore, the aim of this study was to determine if: 1) the number of sperm per dose used in the AI station could be minimized in outstanding bulls without affecting fertility, 2) variations arise among five bulls based on oocyte cleavage rate, 3) different stages of embryonic development affected due to bull differences, and 4) embryos produced as a result of these five bulls exhibit different freeze-thaw survival rates. The results of the study might offer the AI industry a useful, simple and inexpensive laboratory test to evaluate the performance of prospective sires. The processes involved in the IVF system and the possible variations that were comprehended in the system have been reported in chapter two of the literature review section of this thesis. Altogether, two experiments were conducted in this study and each experiment has been treated as a separate chapter with an introduction, materials and methods, results, 4 discussion and conclusion. Evaluation of five bull performances in IVF system in terms of their ability to cleave oocytes to the two-cell stage (oocyte cleavage rate) are based on: 1) the bulls' genetic merit and 2) three different levels of sperm concentration for each bull has been reported in chapter three of this thesis. The effect of five bulls in embryonic development beyond cleavage of two cell stage and the embryos produced as a result of different bulls tolerance for freeze-thaw survival rate has been described in chapter four of the thesis. Since these two major experiments were done using two different methods of IVF system for semen treatment (capacitating sperm), a pilot study was conducted to compare between the two methods and their procedure and this has been dealt with briefly in chapter five. Finally, a general conclusion covering the two experiments including the pilot study with a brief look of future directions has been provided in chapter six of this thesis. 5 CHAPTER 2. LITERATURE REVIEW A considerable amount of work has been done during the past decade to improve IVF in farm animals. Offspring have been successfully produced through embryo transfer following IVF in farm animals such as cows, pigs, sheep, goats and horses. More emphasis has been given to cattle than any other farm animals. These accomplishments have not only increased our understanding of the process of fertilization in farm animals but also have left the researchers with more opportunities for probing and optimizing every step involved in IVF. Based on the current information available, the following literature review explains the process, applications and problems involved in the IVF system. 2.1. IN-VITRO FERTILIZATION (IVF) The process of fertilization in a test tube or an in vitro system (IVF) has gained much attention following the first report of the success of live offspring resulting from IVF in rabbits (Chang, 1959). In-vitro fertilization had been attempted in at least 14 mammalian species before 1981 (Brackett et al., 1982) . As a result, live offspring born following embryo transfer was reported for rats (Nicholas, 1933), rabbits (Chang, 1959), mice (Whittingham, 1968), human (Steptoe and Edwards, 1978), and bovine (Brackett et al, 1982). Similarly, successful IVF has been reported for other farm animals such as porcine (pig) by Kvansmicku (1951), caprine (goat) by Hanad (1985) and for ovine (sheep) by Cheng et al., (1986). The initial success in obtaining live offspring by IVF was later extended to other domestic, exotic, avian and aquatic species. 7 In-vitro fertilization has been a valuable tool to improve our understanding of many events such as : oocyte maturation, fertilization, early embryonic development, fertility test, and cryobiology of embryos. In vitro fertilization in domestic animals has received much attention in the past two decades, and considerably more progress was made in cattle than in any other farm animal in the field of reproductive technology (Brackett, 1992). In vitro fertilization in the bovine is in the process of commercialization particularly for reproductive and genetic improvements. In vitro fertilization in the bovine has many advantages such as reducing the generation interval (Kajihara et al. , 1991), overcoming infertility, increasing the reproductive life of the cow (Goto et al, 1990), and preserving endangered cattle breeds (Solti et al, 1992). Even though there are several methods available to measure reproductive efficiency of bulls, recent developments in I V F techniques show that I V F can be used as an ideal tool to assess reproductive performance. The I V F technique may be used as a tool to predict bull performance using oocytes collected from local slaughterhouse ovaries and frozen semen obtained from the AI center. As a result of this effort, the in vitro fertilization technique has contributed many successful results in recent years. 2.2. T H E O V A R I E S A N D T H E O O C Y T E S Ovaries serve as the primary sex organ of the female reproductive system. The production of gametes (the ovum) and the female sex hormones (estrogen and progesterone) are the essential functions of the ovaries. In cows, the ovaries are almond-shaped (oval), and vary in size from about 1.5 - 5 cm in length and 1 - 3 cm in diameter and changes in the shape and size of ovaries occur during the reproductive life of the cow. The surface epithelium also known as the germinal epithelium is believed to be the origin of stock female germ cells 8 (oogonia). Since the cow is a monotocous species, it develops and ovulates one egg during each estrous cycle from the right or left ovary. At the time of ovulation, the oocyte is surrounded by corona radiata (granulosa cells surrounding the potential ovum) and a sticky mass containing other granulosa (cumulus) cells which aid egg pickup by the oviduct. 2.2.1 Oocyte Maturation Process In-Vivo The oocyte maturation is a complex process which is regulated by many factors and mostly dependent on the follicular environment. The process of maturation takes place during the period between the luteinizing hormone (LH) surge and ovulation. During this period, the oocyte undergoes both nuclear and cytoplasmic maturation. According to Mattioli et al., (1991), the transition from the growth phase to the maturation phase of the oocyte is dependent on the presence of an active maturation promoting factor (MPF) in the cytoplasm. The oocyte growth phase is responsible for the second reduction division in which the nucleus proceeds through the anaphase to telophase stage. The first polar body is extruded into the perivitelline space following nuclear maturation. The cytoplasmic maturation starts once more on the resumption of meiosis by releasing MPF which needs about 6-8 h. The inhibition release coincides with loosing of granulosa and other cells around the oocytes as a result of gonadotrophine stimulation. The increase in gonadotrophin concentration during the preovulatory period stimulate, the resumption of meiosis. The final stage of development is the egg maturation in which the circulatory FSH and LH make ready the preovulatory follicle. In the female gametes, two separate cell divisions will occur in separate incidents. The first division is just before ovulation and the second before fertilization. The first division is a meiotic division in which the chromosomal content of the oocytes is reduced by half to 30. Only one cell along with a small polar body is produced instead of four cells. The second 9 meiotic division of the oocyte and the protruding of the second polar body occurs with the penetration of the fertilizing sperm. 2.2.2 In-Vitro Maturat ion ( IVM) The ability of the oocytes to undergo maturation when incubated in vitro was first demonstrated by Edwards (1965). Signs of maturation for in vitro recovered oocytes are expansion of cumulus cells, maturation of the nucleus, expansion of the cytoplasmic membrane, and appearance of the first polar body (Ball et al., 1984). Cumulus expansion is easily recognized under the microscope, and it is an important sign of oocyte maturation under in vitro conditions. During expansion, a disturbance occurs both between adjacent cumulus cells and between cumulus cells and the oocyte. This type of disturbance results in the production of hyaluronic acid by the cumulus cells (Ball, et al., 1982). Hyaluronic acid is thought to aid in fertilization. The cumulus cells also produce energy substrates like pyruvate during maturation (Donahue and Stern, 1968). Oocytes recovered from follicles or oviducts during hormone induced ovulation in vivo provides the means of understanding in vitro fertilization (Barackett et al., 1980). Under in vivo conditions, the expansion of cumulus cells starts after the surge of gonadotrophins before ovulation, and is most likely initiated by FSH (Hensleigh & Hunter, 1983). Addition of FSH, LH and estradiol to in vitro maturation media improves the fertility of the matured oocytes (Fukushima and Fukui, 1985). Oocytes that are unable to form a male pronucleus during fertilization lack a male pronuclear growth factor (Thibault et al, 1975) due to inadequate cytoplasmic maturation. The addition of LH to rat oocyte-cumulus complexes during IVM increased the ability of zygote to facilitate the formation of male pronuclei after sperm penetration (Shalgi et al., 1979). Similar results were observed (Motlik & Fulka, 1981) in to rabbit oocytes when human chorionic gonadotropin (hCG) was supplemented. Similarly, estrus cow serum (ECS) enhances oocyte maturation, assists zygote development up to 2-cell stage and gives better results than fetal calf serum (FCS) (Schellande et al, 1990). The somatic cells surrounding the oocyte help in transporting nutrients into the oocyte, and conveying signals into and out of the oocyte. However, somatic cell supplements such as granulosa cells delay nuclear maturation of oocytes in vitro (Leibfried-Ruteledge et al, 1989), and a high density of oocyte cumulus complexes (OCC) might also delay nuclear maturation of the oocytes. Bovine oviductal epithelial cells (BOEC) can be used as somatic cells supplement (Dunford et al, 1992 ) for maturation and have given better results. The size of the follicle from which the oocytes originate does not influence nuclear maturation (Leibfried-Ruteledge and First, 1979; Fukui and Sakuma, 1980; Grimes and Ireland, 1987); however, recent evidence suggests that oocytes which have not completed their growth do not synthesize adequate amounts of RNA (ribonucleic acid), and fail to undergo early embryonic development (Crozet et al, 1986; Crozet, 1989). Therefore, it is advisable to harvest oocytes from medium-sized follicles of approximately 2-8 mm in diameter (Staigmiller, 1988). Since ovulation occurs approximately 28 h after the LH surge in heifers (Christenon et al, 1974), the maturation process in vitro requires less than 28 h (Shea, et al, 1976). Therefore, to complete meiotic maturation, the oocytes have to be provided with an adequate microenvironment for 18-27 h. The optimum pH of 7.2 - 7.4 might help in oocyte metabolism by influencing the mitochondrial enzyme activity (Biggers, 1972). Therefore, enzymes that play a major role in bovine oocyte maturation might be inactivated above pH 7.6 or below 6.7. The ability of the oocyte to mature in vitro does not depend on the stage of the estrous 11 cycle (Leibfried and First, 1979). However, Sivakumaran, et al, (1993) showed that oocytes obtained from pregnant and cyclic cows underwent higher maturation rate than those from anestrous cows. Similarly, the study conducted by Savio et al, (1988) found that corpus luteum (CL) bearing ovary contains more follicles. The transporting time from slaughterhouse to the laboratory and the optimum temperature for holding ovaries during this period might influence the maturation of oocytes. Other factors like body condition, reproductive status and type of breed also affect the maturation of oocytes. 2.2.3 Harvesting Oocytes From Ovaries For in vitro fertilization, oocytes can be collected surgically and non surgically from live animals or from slaughterhouse ovaries . Surgical techniques have been developed for collection of ova from both laboratory (Daniel, 1971) and farm animals (Dzuik, 1971; Murry, 1978). Oocyte collection from live farm animals was developed in 1980 using laparoscopy (Lambert et ah, 1983,1986) and colpotomy (Stubbings et ah, 1988). Ultrasound guided transvaginal aspiration of oocytes was first performed in cattle in 1987 (Callesen et al., 1987). At present, most laboratories around the world use slaughterhouse ovaries as the source of oocytes. The ovaries can be cut, minced or a needle attached to a syringe can be used to aspirate oocytes from small-size follicles. In the first method, the ovary is immersed in solution and the outer surface is cut into thin layers (Hamanoa et al., 1993) and the oocytes are then recovered from the solution. The aspiration method using a needle attached to a syringe involves recovering oocytes from visible follicles measuring 2-8 mm in diameter on the surface of the ovary. The mincing method is usually done after aspiration to recover more oocytes per ovary (Takagi et ah, 1992). The methods of harvesting oocytes might not affect the end 12 results of IVF; however, the aspiration method is most commonly used because of its simplicity and ease of collection. The average number of oocytes that can be recovered from an ovary using the aspiration technique ranges between 7-10 depending on the reproductive status of the cow. Ultrasound guided transvaginal aspiration of oocytes from cows after superovulation is a promising technique that could find wider application. 2.3 BULL SPERM PREPARATION FOR IVF The development of spermatozoa in mammals is known to occur in two phases. The first phase, spermatocytogenesis, involves a series of cell divisions in which spermatogonia (the stem cell) form spermatids (the haploid germ cell). The second phase, spermiogenesis, comprises the transformation of spermatids to fully organized spermatozoa. Most of the metamorphic changes take place during spermiogenesis including head and tail formation. The acrosome, a cap around the head of the spermatozoa that arises from the Golgi apparatus is also formed during this phase. The whole process of spermatogenesis takes about 56-63 days in bulls. Spermatozoa gain progressive motility and the ability to fertilize the egg following maturation in the epididymis. The source, quality and treatment of semen has a great impact on in-vitro embryo production. Spermatozoa collected from the epididymis were used for IVF after freezing and thawing to produce embryos with successful establishment of pregnancies (Goto et ah, 1988). Freshly collected ejaculates have also been used to produce normal live calves (First and Parrish 1987). Spermatozoa taken from a freshly collected ejaculate have a better quality than epididymal spermatozoa in their ability to produce embryos in vitro ( Pavlok et ah, 1989). Brackett et al., (1982) produced the first bovine calf through IVF using frozen semen. Frozen thawed semen is a more convenient and abundant source of spermatozoa for IVF in order to 13 reduce bull variations. The following section of the review focuses on essential processes which bull spermatozoa have to undergo before they gain the ability to fertilize the egg both in vivo and in vitro. 2.3.1 Capacitation And Acrosome Reaction Of Spermatozoa One of the vital functions of the epididymis is to help in the maturation of spermatozoa. Spermatozoa entering the head region of the epididymis from the seminiferous tubules via the vas efferentia are neither motile nor fertile and gradually gain these abilities during their passage from the body to the tail region of the epididymis. The spermatozoa lose again the fertilizing potential when seminal plasma is added during ejaculation. The spermatozoa become fertile once more if seminal plasma is removed. Therefore, there are two processes of maturation which occurs in spermatozoa before fertilization. The first maturation occur in the epididymis as discussed above in gaining the ability to be motile and fertile. The second maturation is independent from the male reproductive system and occurs in the female reproductive tract. It is believed that mammalian spermatozoa must spend adequate time in the female genital tract removing the contents of seminal plasma to fertilize the egg (Austin, 1951; Chang 1951). During this period, some physiological and biochemical changes allow the spermatozoa to penetrate the egg. These processes were defined for the first time by Austin (1951) and are called capacitation. Capacitation has been defined as cellular changes that spermatozoa undergo in female reproductive tract that are necessary before acrosome reaction and fertilization can occur. The precise change involved in capacitation are not fully understood, but they involve enzymatic and structural modification to the acrosome and anterior part of the sperm head membrane. These include: 1) an increase in membrane permeability to calcium 2) modification of the membrane structure 3) activation of the enzyme 14 adenyl cyclase 4) conversion of the protein proacrosin to acrosin (Clegg, 1983). This process of maturation involves, removal of cholesterol from sperm plasma membrane, removal of glycoprotein from the head region, hyperactivating the motile sperm and an influx of calcium. The agents causing capacitation and the mechanism involved in capacitation have been poorly understood; however, several substances such as heparin (Parrish et al., 1986), caffeine (Goto et al., 1988), lysolipid and trypsin (Wheeler and Seidel, 1986) have been used routinely to capacitate bull spermatozoa in vitro. Heparin belongs to a group of polysaccharides called glycosaminoglycans (GAG) and has been used widely for capacitating sperm in vitro (Parrish et ah, 1986). Heparin gives better results in sperm penetration of oocytes if added for 4 h in the case of fresh semen and 15 min. for freeze-thaw semen prior to oocyte exposure (Parrish et al., 1988). The concentration of heparin to be added differs between bulls and also depends on the type of semen used (Leibfried-Ruteledge et al, 1985). Glycosaminoglycans (GAG) are also helpful in changing the membrane of the acrosomal cap during acrosome reaction. Capacitation and acrosome reaction are very closely linked, and therefore, it is not always possible to differentiate the two processes (Clegg, 1983). Acrosome reaction (AR) involves fusion of the outer acrosomal membrane with the plasma membrane of the spermatozoa leading to the formation of vesicles followed by release of proteolytic enzymes needed for sperm penetration of the zona pellucida. Acrosome reaction (AR) helps the spermatozoa to penetrate the zona pellucida and allows the fusion of sperm surface with the oocyte plasma membrane. Glycosaminoglycans (GAG) do not induce acrosome reaction, but they do prepare the spermatozoa to respond to calcium (Hanada, 1985) since calcium influx is a vital requirement for acrosome reaction. 15 Caffeine, a naturally occurring alkaloid has also been used for inducing sperm capacitation in vitro. Caffeine is believed to stimulate motility, respiration and fructolysis of both epidermal (Fattouh and Abdou, 1991) and ejaculated spermatozoa (Garbers et al, 1971). This finding in cattle is supported by Schoenfeld et al, (1975) and for human spermatozoa by Barkay et al., (1977). In conjunction with other capacitation agents , it is assumed that caffeine might improve fertilization rates in vitro by acting as an inhibitor of phosphodiesterases resulting in an increase in cAMP levels in spermatozoa (Brackett and Zuelke, 1993). Capacitating freshly collected spermatozoa using high ionic strength solutions (HIS) has also resulted in the production of calves following IVF (Sirard & Lambert, 1985). Similarly, ionophores, xanthine derivatives, and somatic cells such as oviductal epithelial cells and cumulus cells have been used for capacitating spermatozoa. 2.4 FERTILIZATION, CLEAVAGE AND EMBRYONIC DEVELOPMENT The process in which the haploid male and female gametes unite in the formation of the diploid zygote is called fertilization. In most species, the ovum is in metaphase II of the second meiotic division during ovulation. Maturation and meosis are completed after fertilization when the ovum becomes a zygote. The initial step in fertilization involves the penetration of the spermatozoa through the cumulus and corona radiata cells. This penetration from the vitelline membrane to the cytoplasm of the egg is aided by enzymatic process and the phagocyte action of the spermatozoa (Yanagimachi, 1988). During fertilization, penetration of sperm through zona pellucida activates the egg and the zona pellucida becomes hardened. Following the penetration of spermatozoon into the zona pellucida to the perivitelline space, spermtoaozoon fuse with the plasma membrane of the oocyte. The zona pellucida, the, glycoprotein coat of the oocyte, prevent the entry of other spermatozoa. As a result of fusion 16 with egg plasma membrane, the spermatozoon nuclear envelope disintegrates and forms the male pronucleus. The second meiotic division is resumed by the formation of the second polar body of the oocyte and to the perivitelline space followed by the shrinking of the cytoplasm. Both pronuclei migrate to the center of the oocyte where the nuclear envelopes disintegrate and the chromosome of the male and female pronuclei combine to form the prophase of the first mitotic division. The aggregation of chromosomes in the prophase of first cleavage division results in the formation of the zygote in the diploid state. The intermixing of the chromosomes might be considered the end of fertilization and start of embryonic development (Parrish and First, 1991). The start of the first mitotic division in the zygote after fertilization but without growth and enlargement of the cytoplasm is called cleavage (Beaden and Fuquay, 1992 ). One of the standard evaluation procedure used in cattle IVF is the percentage of oocytes that have cleaved after two days of incubation. The first cleavage that results in a 2-cell embryo under in vivo conditions takes about 24 h and takes between 20-36 h in vitro. As the mitotic divisions continue, embryonic development also proceeds simultaneously. Further cleavage results in the formation of 4, 8, 16-cell, embryos up to the blastocyst stage. Once the cell reaches the 16-32 cell stage, the structural form makes cell counting difficult. In the live cows, four cell stage, 8-cell and morula stage are reached at about 42, 58 h and 82 h respectively. On the other hand, the rate of embryonic development in vitro study was inconsistent. The time required for embryonic development in vitro for two cell, four cell, eight cell, morula and blastocyst stages varies from 20-36 h., 48-52 h., 72-90 h., 144-170 h. and 168-216 h, respectively. At the end of cleavage, the zona pellucida breaks and the blastocyst is released. The process of blastocyst release from the zona (hatching) takes about 14-16 days in vivo and 9-12 days in 17 vitro (Massip and Mulnard, 1980). The free blastocyst undergoes further elongation. Cleavage ends with the formation of the elongated blastocyst and further cell divisions continue with growth. 2.5 FACTORS AFFECTING CULTURE OF SPERM AND OOCYTES Successful embryo production following fertilization and cleavage is affected by several factors such as oocyte quality, oocyte maturation method, semen quality, semen preparation, media used for culture of sperm and egg, coculture system used, and other factors ( Hyttel et al., 1991a; McCaffery et al., 1991). All factors that affect the oocyte maturation process affect oocyte quality. Oocytes with a translucent appearance generally have a lower potential for fertilization. Oocytes free from any debris, morphologically tight and complete with multilayered cumulus investment undergo normal fertilization. Yang & Lu (1990) showed that oocytes denuded of cumulus cells gave lower cleavage rates than oocytes with intact cumulus cells following IVF. The supplementation of somatic cells during fertilization affects both fertilization and cleavage rate of oocytes. Leibfried-Ruteledge et al., (1989) found that the presence of granulosa cells during IVF lowers the fertilizeability of oocytes. On the other hand, the addition of cumulus cells during IVF has increased the fertilization rate of oocytes (Shamsuddin and Larsson, 1993). An optimum temperature of 39 °C has been shown to give better fertilization rates. The duration of sperm-egg incubation also has an effect on in-vitro fertilization method. The shorter the time of incubation, the lesser the incidence of polyspermy. 18 2.6 IN-VITRO CULTURE (IVC) In vitro matured and fertilized (IVM-IVF produced) embryos of farm animals rarely reached the blastocyst stage due to blockage either at the 4-cell (Barnes & Eyestone 1990) or the 8-16-cell stage ( Camous et al, 1984). To overcome this developmental block, bovine embryos were cultured either in rabbit or sheep oviducts embedded in agar (Elsden et al, 1982) or with somatic cells (Eyestone et al., 1987). However, embryos incubated in rabbit oviduct were mostly lost or did not develop at a normal rate (Elsden et al., 1982). As this developmental block remained unresolved, the use of somatic cells in the in-vitro culture system has become a routine procedure in most laboratories. Bovine zygotes have been successfully cocultured to the blastocyst stage in a relatively simple media supplemented with either serum or bovine serum albumin (BSA) (McLaughlin et al., 1990). Growth factors such as transforming growth factor (TGF-(3) have also been used in coculture. 2.6.1 Co-Culture With Somatic Cells The use of somatic cells, HeLa cells, and other cells for coculturing mouse embryos was first demonstrated by Cole and Paul (1965). They found that these cells might provide energy sources like lactate or pyruvate for the developing embryo. Fibroblast cells obtained from bovine endometrial tissue, 4-5 d after the onset of estrus were also used to culture bovine embryos (Frank and Raymond, 1982) resulting with successful hatching of the blastocyst. They hypothesized that these cells either released a "factor" that promotes embryo development or had the ability to remove toxic substances from-the media. The presence of releasing factors in somatic cells is supported by the presence of peptides, hormones and other factors in cell monolayers (Allen and Raymond, 1984). Bovine oviductal epithelial cells (BOEC) supplemented without or with 10-20% ECS (Fukui and Ono, 1988b), granulosa cells 19 (Goto et al, 1988) and trophoblastic vesicles (Aoyagi et al, 1988) have been used for culturing embryos. 2.7 MEDIA FOR IVF In order to achieve successful fertilization in vitro, the development of suitable culture systems and media has become essential. Several media have been used since the beginning of IVF and the improvement is still in progress (Menezo and Khatchadourian, 1991). During the early stages, culture media were prepared by the addition of vitamins, amino acids, nuclear bases, various macromolecules, serum and various growth factors. The following section deals with some useful information currently available on laboratory culture media. 2.7.1. Inorganic Ions, Organic Compounds And Sera For Culture Media The major inorganic ions used in preparing culture media are cations such as sodium (Na), potassium (K), magnesium (Mg), and calcium (Ca), and anions such as chlorine (Cl), carbonate (HC03), sulphate (S04), and Phosphate (P04). The concentration of these inorganic ions used in the culture media differ among laboratories. The mouse embryo usually cease to develop beyond 2-cell stage if the concentration of sodium in the media is high (Lawitts and Biggers, 1991). Adequate amounts of potassium in culture media on the other hand helps in sperm-egg fusion, fertilizeability and prolonging the life span of the spermatozoa in mice (Boldt et al, 1991). Even though the presence of Mg, S0 4 and P0 4 in the media is thought to be important, these chemicals might as well inhibit or impair embryonic development (Wales, 1970). Some of the beneficial effect of adding bicarbonate (HC03) in the media are as follows: acts as a buffering agent in the media, helps in activating the spermatozoa and also acts as a source of carbon for embryo metabolism (Quinn and Wales, 20 1973). The quality of water used also affects embryonic development due to the presence of other inorganic compounds such as zinc, copper, manganese, and other chemicals which affects embryonic development; however, the concentration of these chemicals is heavily dependent on the quality of water used for IVF. Considerable variation exists in the composition of various organic compounds used in preparing the culture media. These compounds are either supplied as saline such as Tyrode's, Locke's, Whitten's, Earles's or biological fluids such as Ham's F-10, Ham's F-12, and TCM 199. Commonly employed organic compounds in culture media are 1) energy substrates such as glucose, pyruvate and lactate 2) lipid sources such as bovine serum albumin and 3) amino acids and proteins (Fallon et al., 1988). The addition of serum in the media serves as a source of enzymes, some growth factors and lipids. Although there are several advantages in adding serum, the adverse effects of some proteolytic enzymes that may be present in serum has to be investigated (Menezo and Khatchadourian, 1991). Overall, the efficient use of culture media is further influenced by other biophysical factors such as osmolarity, pH and the gaseous phase used in the in vitro system. 2.8 MEASUREMENT OF REPRODUCTIVE EFFICIENCY Reproduction is a critical factor in determining the efficiency of animal production. Therefore reproductive efficiency can be described as a measure of the ability of a cow to become pregnant and produce potential offspring (Peters and Ball, 1987). Infertility or sub-fertility are varying degrees of irregularity from typical levels of reproductive performance. Most likely a cow produces only a single calf per year. As a result the genetic progress of 21 bovine reproduction is very slow and less efficient than other farm animal species. The better the management, the better is the reproductive efficiency in farm animals. Reproductive efficiency in farm animals is a guideline to determine the ability of both males and females to produce an offspring or to replace the herd for economic purposes. Reproductive efficiency is usually expressed in percentage and is determined in the field using certain other guidelines some of which are briefly summarized below. 2.8.1 Non return rate (NRR) As the importance of artificial insemination (Al) increased, the need to evaluate or to assess the performance of the bull within a short period became necessary. In order to fulfill this task, the Al industry began to calculate the non return rate (NRR) of cows following insemination. NRR is defined as the percentage of cows in the herd that do not return to estrus or those do not receive a second service within a designated time interval. The most commonly used time intervals are 28 to 35 days, 60 to 90 days and 150 to 180 days. Longer time intervals provide more accurate information, yet shorter time intervals are beneficial since the results are available early. Although NRR is influenced by both male and female factors, this method has been widely accepted to evaluate bull performance by the Al industry . 2.8.2 Calving rate From a biological point of view the calving rate is the most appropriate measure of fertility. This is defined as the number of calves born per 100 services (Peters and Ball, 1987). The calving rate is calculated by dividing the number of calves born by the total number of 22 cows in the herd and is expressed in percentage. It can also be expressed as net calf crop and is calculated as the total calves weaned by the total cows in the herd. 2.8.3 Calving interval Fertility is usually estimated based from an economical point of view. Therefore, the calving interval is defined as the period between successive calvings (Berger et al., 1981) . For simplicity, the calving interval can be divided into the calving to conception interval and the gestation period. The calving to conception interval is the time from parturition until the establishment of the next pregnancy. The gestation period is normally between 280 and 285 days in cow. This is influenced by both male and female genetic factors. 2.8.4 Services Per Conception The number of services per conception is determined in a herd or flock by dividing the number of animals serviced by the total number of animals that conceived and is expressed in percentage. This measurement is valid for cows (Berger et al., 1981; Stevenson et al., 1983). 2.9 EVALUATION OF BULL FERTILITY IN VITRO The ability of the sperm to fertilize an egg following insemination is a definite means to predict fertility (Diskin, 1987). The ideal method to test bull performance was based on the ability of the bull to induce pregnancy and subsequent birth of a normal offspring following insemination (Bavister, 1990). Therefore, a reliable and inexpensive laboratory test to predict performance of the bull following insemination will be of great value for both research and economic purposes. Routine semen evaluation manipulates several parameters to determine sperm quality as a measure of bull performance. The following criteria are some of the methods used in determining fertility: 23 2.9.1 Consecutive Sperm Motility The percentage of sperm cells that move progressively from one point to another in a more or less straight line under their own competence is called motility. The percentage of motile sperm is usually determined under a microscope, and for ejaculated semen the percentage ranges from 0 - 80%. It was found that a semen sample that has less than 40 % motile sperm is not suitable for fertilization. Recently a computer automated semen analyzer has been used to evaluate motility. With the help of this computer system, Budworth et al, (1988) found a relationship between fertility and motility for bull spermatozoa. Nevertheless, subsequent studies done by Bailey et al., (1994) found no correlation between computer evaluated motility and in vivo fertility of frozen bull semen. 2.9.2 Sperm Cell Morphology The normal spermatozoon can be structurally divided into head, mid-piece and tail. Sperm head consists the important components for a genetic code. Several studies have shown conflicting results between morphology and fertility ranging from zero to high percentage Saacke (1982). However, lack of motility or presence of an abnormality in the spermatozoa might lead the cell to be infertile. Even though abnormal spermatozoa in ejaculated semen range from 5-100 %, fertility is usually compromised only if the percentage of abnormal spermatozoa exceeds 20-25 %. 2.9.3 Staining of Live and Dead Sperm The live sperm percentage is always higher than the motile or abnormal sperm percentage; therefore, it is important to use a reasonable technique to evaluate the live and dead spermatozoa. For example, Eosin is a differential stain that can pass through a non living 24 cell but not through a living cell membrane due to the intact plasma membrane. A background stain such as nigrosin, opal blue or fast green makes the unstained spermatozoa head visible. The correlation of live spermatozoa and fertility potential of spermatozoa is well established (Hafez, 1993). 2.9.4 Other Tests Many other parameters have been suggested and employed to evaluate and test the semen sample for fertility. The concentration of spermatozoa, resistance of sperm to cold shock, metabolic activity such as oxygen uptake, fructolysis, pH change, and other factors have been used to determine bull semen fertility and need more investigation (Mann, 1975; Hafez, 1993). 2.9.5 The Relationship Between In Vitro And In Vivo Fertility Of Bulls The IVF technology has gained much attentions in focusing beyond embryo production by employing other research applications. One of the interesting attempts is the establishment of relationship between the 60-90 d NRR and the in vitro results for successful pregnancy. The chances of obtaining a definite relationship is not only conflicting among researchers but also the repeatability varies tremendously (Shamsuddin and Larsson, 1993; ; Shi et al, 1991; Marquant-Le Guienne et al, 1990; Hillery et al, 1990; Iritani et al, 1989; Eyestone and First, 1989; Ohgoda et al, 1988). The study conducted in our laboratory (Ambrose, 1995) supports the existence of a correlation between mAb HS-11 binding to bull sperm for different bulls and the cleavage rate of oocytes following IVF. However the result did not establish any correlation with the in vivo fertility test. Since the study was conducted to predict male fertility in vitro using anti-sperm monoclonal antibodies as biomarkers, it is necessary to 25 establish a reasonable method to test differences in bulls in vitro system using the IVF technique. Since the male specific influence has gained more emphasis in IVF, the relationship between the in-vitro and the in-vivo fertility of bulls might be more interesting area for further investigations (Brackett., 1992). 2.10 THE FREEZING AND THAWING OF MAMMALIAN EMBRYOS Cryobiology deals with the preservation of living matter at very low temperatures. The biophysical applications of cryobiology apply not only for living cells and tissues but also for embryos (Hafez, 1993). Mammalian embryos can be preserved for long periods in a normal state by cryopreserving them at very low temperatures without manifestation of further metabolic activity in the cell (Niemann, 1990). The first successful cryopreservation of bovine embryos was reported by Wilmut and Rowson (1973) and cryopreserved bovine embryos have been successfully transported over long distances (from New Zealand to Australia by Bilton and Moore, 1976a, 1976b) The advantages of preserving mammalian embryos can be listed as follows: embryos can be transferred to a foster mother without the risk of genetic changes; animal breeding centers can store surplus stocks for future use; space and money can be saved as well protection against loss through natural catastrophes and human errors such as fire, disease and other hazards can be provided; inbred strains, mutations and special genetic combinations; establish genetic pedigree standards and check for genetic drift in subsequent generations can be preserved (Fahning, 1986; Hafez, 1993). For example, cryopreservation of both sheep and goat embryos may serve as a good model for preserving embryos from other small ruminants that are in danger of becoming extinct (Fahning and Garcia, 1992). It is also inexpensive to transport frozen embryos. 26 Although cryopreservation of different mammalian embryos has been attempted, the success rate is variable because of 1) the varied response of the embryo at different stages of development , and 2) the effect of various biophysical and physiochemical factors such as cooling media, nature and concentration of the preserver, type of freezer used and the thawing rate. The following section deals with the major applications and steps involved in cattle embryo preservation. 2.10.1 Principles Of Cryopreservation The principle of cryopreservation is to get rid of as much water as possible from the embryo. Consequently, the removal of water from the inner cell prevents intracellular ice formation. The mechanism of permeability of the cell helps in keeping the cytoplasm supercooled until freezing. When a cell is cooled below 0 °C, ice crystals form outside the cell. As a result, the concentration of solutes (the substances dissolved in the solution) in the remaining water increases. It was suggested that the rate of freezing (either slow or fast) will affect embryo freezing due to ice crystal formation. According to the principle of osmotic pressure, the cell acts as a barrier to prevent the spread of ice crystals into the intracellular compartment. The formation of large intracellular ice crystals may occur during cryopreservation due to increased intracellular concentration of solutes or dehydration of cells due to solution effects (Fanning and Garcia, 1992). Hence, fast freezing minimizes damage from solution effects, but it leads to the formation of large ice crystals which may cause severe mechanical damage (Trounson et al.,\916). On the other hand, slow freezing prevents large ice crystal formation, but it leads to increased damage from the solution effects. Therefore, the optimal freezing rate of cells or tissues depends on the ability to withstand the damage from ice crystal formation and toxicity from the solution. 27 If the cell is sufficiently permeable to water, pressure remains low and dehydration results as water moves out of the cell to freeze extracellularly. The extracellular ice crystals deform the cell by rupturing the plasma membrane. This cell deformity can be prevented by the addition of cryoprotectants (usually they penetrate the cells) such as glycerol, 1,2-propanediol (PROH), and ethanol and dimethyl sulfoxide. Before freezing, embryos are exposed and equilibrated to cryoprotectants since cryoprotectants penetrate the cells. During the exposure, embryos start losing water due to hyperosmoticity of the exracellular solution considering the embryos are more permeable to water than to the Cryoprotectants. Shrinkage stops when equilibrium is reached between the efflux of water and the influx of cryoprotectant (Schnider, 1986). The embryos reexpand after attaining equilibrium. These Cryoprotectants reduce dehydration of cells and thereby prevent harmful solution effects (Suzuki et ah, 1990; Schneider and Mazur, 1984). When these cryoprotectants are added to the freezing medium, the medium can be frozen at lower temperatures. Prior to freezing to very lower temperatures, a holding period of 5 to 10 min. is necessary to equilibrate both temperature and cell volume. This is done by artificially induced crystallization (seeding) at -6 to -7°C at 10 °C/min (Niemann, 1990; Leibo, 1990). Seeding induces a phase change from water to ice that helps increase the concentration of the salt in the solution (Leibo, 1985). The rate at which the embryos are cooled might effect the process of cryopreservation. A slow cooling produces a gradual shrinkage as water flows out of the cells and freezes outside the cell. It has been shown (Tervit and Elsden, 1981) that the volume of embryos was reduced by 50 % and 40% at temperatures 15 °C and 20 °C respectively. On the other hand, rapid cooling allows freezing of the cytoplasm. The embryo cooling rate depends on surface to volume ratio (surface area of the cell to the volume of solution), temperature, 28 hydraulic conductivity or the water permeability coefficient and the degree of the cooling temperature for water permeability (Leibo, 1989). Slow cooling will be done at a rate of 0.3 to 0.1 °C /min. to -60 to -120 °C at which stage the embryos are plunged and stored in liquid nitrogen (-196 °C ) to obtain extensive dehydration. The sudden freezing to a lower temperature or thawing to a higher temperature will reduce the viability of the cell. Since embryos have low permeability to water, it has been suggested cooling very slowly at a rate below 1 °C / min. and rewarmed at 4-25 °C7 min (Fahing and Garcia, 1992) helps in the process of cryopreservation.. During thawing of cells, the adverse effects of intracellular ice crystals are caused by their growth and recrystallization. As a result, osmotic stress could be imposed on cells when intracellular ice melts. Therefore, warming at a desirable rate ( 4-25 °C/min. ) will dehydrate or rehydrate the cell without affecting the original viability of the cell (Takeda, 1987; Willadsen et al, 1978). 2.10.2 Use of Cryoprotectants Cryoprotective agents are organic solutes which protect cellular organelles during cryopreservation. The addition of a cryoprotectant is necessary to avoid damage to the embryo during freezing as well as thawing. The two major types of cryoprotectants are: penetrating cryoprotectants such as glycerol, dimethylsulfoxide (DMSO), 1,2-propanediol (PROH), ethanol, and non-penetrating ones such as polyvinylpyrrolidone (PVP), sucrose, glucose or other sugars. (Niemann, 1985). Even though the shrinkage and subsequent expansion of the cell may occur during the addition of cryoprotectants, these features are the indications of the response for osmotic changes to hyperosmotic environment (Niemann, 1990). Recently it was shown that 1.6 M, of PROH results in high survival rates for both in vitro and in vivo produced embryos (Suzuki etal, 1990). 29 2.10.3 Controlled Freezing and Thawing The majority of bovine embryos have been cyropreserved using controlled freezing and thawing methods (Niemann, 1991). However, there are other methods of freezing embryos which could be helpful in the future such as one-step, vitrification, and ultrarapid freezing. 2.10.4 Media Supplementation Dulbecco's phosphate buffered saline (PBS) supplemented with either serum or BSA has been widely used as the basic medium before, during and after both freezing and thawing embryos (Seidel et al, 1990). However, during shipment of the embryos, it is necessary to replace sera or BSA by defined macromolecules in order to reduce the possibility of disease transmission (Seidel et al., 1990). Further investigation is necessary to attain optimal medium composition, in particular the protein source for embryo freezing in order to prevent disease transmission (Niemann, 1991). 2.10.5 Loading Embryos into Straws And The Freezing Machine Embryos are usually loaded into straws of 0.25 ml capacity. These straws are better when compared to ampoules or plastic tubes since a thinner wall, smaller volume and smaller diameter makes not only the seeding process faster and more accurate but also improves survivability. Type of straws from various manufactures with regard to evaporation of ethylene oxide after gas sterilization has a considerable effect on embryo survival (Schiewe et al., 1990). Therefore, selecting the right type of straws is a crucial factor in freezing embryos (Whittingham, 1971). Several manufacturers offer sophisticated and expensive machines that allow different freezing and thawing programs. In this experiment we used Bio cool II FTS® system. While using ethanol freezers it is necessary to maintain the level of alcohol with time in order to avoid 30 change in freezing rate and contamination with water as a result of condensation. Using pure ethanol (99.6% pure) for embryo freezing prevents alteration of the freezing rate (Niemann, 1991). 31 CHAPTER 3 . BULLS AND SPERM CONCENTRATION EFFECT ON BOVINE OOCYTES CLEAVAGE RATE 32-CHAPTER 3 BULLS AND SPERM CONCENTRATION EFFECT ON BOVINE OOCYTESCLEAVAGE RATE 3.1 ABSTRACT This study was conducted to examine if differences among bulls and sperm concentrations used for IVF affect bovine oocyte cleavage rate, with the ultimate objective of deterrnining whether sperm number per dose could be reduced in outstanding bulls without affecting fertility in vivo. The cleavage rate of bovine oocytes subjected to IVF using three different sperm concentrations (2xl06, lxlO6 or 0.5xl06/ml) was assessed. Frozen-thawed semen samples of five Holstein bulls were tested. Cumulus oocyte complexes aspirated from 2-8 mm follicles (slaughter house ovaries) were matured in vitro in Hams F-10 medium supplemented with 10% estrus cow serum for 24-26 h. In vitro matured oocytes were randomly allocated for fertilization to microdrops containing either 200,000, 100,000 or 50,000 total sperm (representing the 2xl06, lxlO6 or 0.5xl06/ml concentrations respectively) in IVF-TALP medium (Parrish et al, 1988) with bovine oviductal cells. Cleavage of zygote to 2-cell stage and beyond was observed and recorded 56-72 h post insemination. Approximately 150 oocytes were used per bull (n=728). Cleavage rates induced by spermatozoa of the five bulls (# 257, # 386 , # 391, # 398, and # 438) tested, were 29.2, 43.2, 36.5, 34.7 and 62.8% (±4 .1% SEM) respectively. Spermatozoa of bull # 438 yielded a high cleavage rate which was significantly different (P< 0.001) from the others. Bull 386 was significantly different (P<0.05) from bull 257 in terms of cleavage rate, but was not different from bull 391 and 398. For the three sperm concentrations of 2xl06, lxlO6 or 0.5xl06/ml, the pooled results of oocyte cleavage rates were 44.5+3.2, 42.0+3.2 and 33 37.4+3.2% (P>0.05) respectively. Further, differences in sperm concentration within a bull did not affect the cleavage rate (P>0.05). Results demonstrate that spermatozoa of different bulls have different abilities to fertilize oocytes in vitro and that even a low concentration of 0.5x106 sperm/ml might be sufficient for successful fertilization and cleavage. The in vivo fertility estimates of the bulls based on 60-90 d non return rate to first insemination were not correlated (r=0.51; P>0.05) with the fertility estimates based on in vitro cleavage. 3.2 INTRODUCTION Bovine IVF techniques have progressed rapidly in the past few years ( Brackett and Zuelke 1993) leading to commercialization of the technique for in vitro embryo production (Greve et al., 1993 ). The recent development in ultrasound guided oocyte aspiration in combination with IVF allows production of embryos from known donor cows and bulls of high genetic merit. Commercial semen straws of 0.25 ml capacity containing approximately 20xl06 cells has been found to be optimal for artificial insemination (Al), based on field insemination data (Greve et al., 1993). However, early studies show that optimum conception rates can still be achieved with low sperm concentration. Kim and Lee, (1972) obtained up to 61% non return rate (NRR) at 60-90 days post Al , using 2xl06 sperm cells. Similarly, in a fertility trial conducted by Jondet (1972) , NRR of 72.5 % obtained from 3xl06 post thaw live sperm cells was identical to control doses of 14xl06 sperm cells. Unlike in the in vivo system, for IVF, a concentration of less than 5xl06 sperm/ml has been used conventionally. Since 20-30 oocytes could be fertilized in a microdrop of about 100 ul, the number of sperm required per drop for fertilization is often in the range of 200,000 to 500,000. As the selection of highly active spermatozoa (either by swim-up separation or other means) has not been found to be an essential 34 requirement for successful fertilization and embryo production , spermatozoa contained in one straw would be sufficient to fertilize up to 300 oocytes in vitro, assuming an inseminate of 200,000 sperm per IVF drop. Frozen semen straws, particularly from superior bulls, are priced several times higher than low fertile bulls in the AI industry. From the foregoing reports it is clear that optimal conception rates can be achieved with a minimum concentration of about 2 - 4 xlO 6 cell /ml (Madison et al, 1991, Vergos, 1990). Normally results from field reports are obtained only after 60-90 days post AI. Therefore, if results can be generated in a short time span, more bulls could be evaluated for their optimal sperm numbers required for fertilization. The objective of the present study, therefore, was to investigate a) if low sperm concentration (< 200,000 sperm per inseminate) would affect bovine oocyte cleavage in vitro and b) if spermatozoa from different donor-bulls affects differently cleavage in vitro. One such protocol in our laboratory uses IVF as a tool to select superior bulls based on performance as prospective semen donors and to determine if sperm per dose could be reduced in outstanding bulls without affecting fertility in vivo. By doing this, the number of semen doses obtainable from superior donors per ejaculate could be increased , thereby cutting down the cost of semen. The routine motility and density evaluation of semen after collection from the bull in AI centers determines whether the semen is accepted or rejected for use in AI. As a rule, semen showing low initial progressive motility or low sperm density is normally rejected in the center. Similarly, semen characteristics that are often associated with fertility levels shown by bulls in NRR results in the field has been used to evaluate bull performance. In spite of achievement have been claimed in satisfactory results after semen evaluation and NRR field data, there are many incidents in which unproductive fertility rates occur. Therefore, the search for other efficient method for predictions of bull performance 35 are needed to reduce expense and time. It was suggested by Anderson et al, (1992) that the use of IVF to predict fertility would save time and expense in comparison with NRR. 3.3 MATERIALS AND METHODS 3.3.1 In Vitro Oocyte Maturation: In vitro maturation of oocytes involves the collecting of ovaries, aspiration of oocytes from ovaries and induction of maturation by artificial means out side the reproductive tract of the cow. The process of maturation involves a variety of media, media supplements, hormones, serum and somatic cells addition to enhance the process, (see appendix C for the step wise procedure of IVF) 3.3.1.1 Collection of ovaries Ovaries were collected from cows and heifers immediately after slaughter in a local slaughter house. Healthy ovaries were placed in 0.9% (w/v) physiological saline (NaCl) at 30 - 38 °C in a thermos flask and transported to the laboratory within 3 hours from the time of collection. The ovaries were rinsed 2-3 times using physiological saline before oocyte aspiration. 3.3.1.2 Oocyte aspiration Ovaries were soaked in the same saline in a large glass beaker and left inside a water bath (at 37 °C) before aspiration. Ham's F-10 medium (Gibco) was supplemented with 10% (v/v) estrus cow serum (ECS) and adjusted to pH 7.35 - 7.40. The estrus cow serum (ECS) was collected from a cow in estrus. The collected serum was heat inactivated. The medium was filtered through 0.2 um filter (Baxter Canlab, Toronto , Ont. ), and used for both aspiration and maturation of the oocytes. Oocytes were aspirated from 2-8 mm size follicles with an 18-gauge needle attached to a 10 cc syringe (Luer Lok, Rutherford, New Jersey, USA) 36 which contained 1 cc of media. After washing 2-3 times with the media, oocytes with multilayered compact (3-4 cell layers) cumulus oocyte complex (COC's) were selected from the aspirated fluid under a dissecting microscope. The selected COC's were washed 4 - 5 times using the same media in a 35 mm dish (Falcon 1008, Lincoln Park, N.J.). Selected oocytes with a uniform cytoplasmic membrane were placed in a 90-p.l drop of maturation medium under 1 ml of sterile light mineral oil (Sigma, USA) , which was distributed in a 4-well multidish (Nunclon Delta, Denmark) which had been pre equilibrated at 39 °C, 5% C0 2 for two hours inside the incubator and then the oocytes cultured at 39 °C, 5% C 0 2 for 24 - 26 hours. 3.3.2 Oviductal cell preparation: Oviducts were collected from the slaughter house at the same time as the ovaries. Oviducts were attached from ovaries bearing large dominant follicles and /or lacked the presence of a corpus luteum. The oviducts were rinsed and trimmed at the frmbrial end. The oviduct was cut at the presumptive ampullary-isthmic junction, and the ampullary portion was gently squeezed with forceps to expel the epithelial cells from the lumen. The cells were collected into a sterile 15 ml centrifuge tube (Corning Incorporated, corning, NY) and repeatedly aspirated with 18-G needle attached with 1 ml syringe to break up the sheets of epithelial cells. After adding 5-7 ml of the media (Ham's F-10 with 10% ECS), the cell were centrifuged for 4-5 minutes at 200 g. The supernatant was removed along with any blood cells. The pellet was resuspended and washed again with the same media up to 4 times. After the final wash the oviductal cells were placed in 35 mm dishes and incubated in Ham's F-10 with 10% ECS at 39 °C and 5% C 0 2 for 24 hours. 37 3.3.3 Semen Preparation Frozen semen from five Holstein bulls (bull # 257, 386, 391, 398, and 438) were used in this experiment. Semen samples from 2 or 3 bulls were randomly allocated for rVF in each experiment. Semen straws were thawed (40 sec in 38 °C water bath) and washed in Sperm-T A L P medium (Parrish et al, 1988) by centrifugation for 5 min. at 200 g. The supernatant was discarded. The pellet of sperm cells was resuspended in sperm-TALP and washed twice more. After the final wash, the supernatant was removed, sperm resuspended in I V F - T A L P and the concentration adjusted to: 2 x 106/ ml (A), 1 X10 6 / ml (B), 0.5 X 106/ ml (C). Spermatozoa of each concentration (A, B, and C) were distributed in 80 ul microdrops and covered with 1 ml mineral oil. The dish was incubated at 39 ° C and 5% C 0 2 for 1 hour before adding the mature oocytes. 3.3.4 In vitro Fertilization: Oocytes with expanded cumulus cells and uniform cytoplasm were removed from the maturation drops after 24-26 h, washed 6 times in H E P E S - T A L P , and 2 times in I V F - T A L P . While washing the oocytes, the cumulus cells were partially removed by repeated aspiration with a micropipette. The oocytes, in 10 ul medium were transferred to the TVF drops to make the total volume 90 ul . Not more than 20 oocytes were randomly added to each drop (A, B or C). Approximately 150 oocytes per bull were used in four replicate trials. After 24 h in culture, oviductal cells were washed 3 times in H E P E S T A L P and 2 times in I V F T A L P . The cells displaying high motility were selected and placed in the I V F drops with approximately 10 ul medium to make the total volume 100 uT The remaining oviductal cells were matured in culture for post I V F procedures. 38 3 . 3 . 5 Assessing Cleavage and in vitro culture Approximately 10 - 12 h after fertilization, loosely adherent sperm cells were removed from the presumptive zygote by gentle rinsing in Ham's F-10 medium supplemented with 10 % fetal bovine serum (FBS). Zygotes removed from the three drops (A, B, or C) were separately cultured with oviductal cells for further development. Cleavage rate was determined by observing the development of the zygote to two-daughter cells (blastomeres) over a given period of time (Plate 3.1). The cleavage rate from each well (A, B, or C) was determined for each bull 56 - 72 h after the time of fertilization. Uncleaved oocytes were removed from culture. 39 PLATE 3.1 CLEAVAGE OF THE ZYGOTE TWO-DAUGHTER CELLS Two-daughter cells (blastomeres) after 24 to 36 h from the time of insemination. A) magnified 200 x B) magnified 100 x 40 3.3.6 Non return rate result The field fertility data obtained by the British Columbia Artificial Insemination Center, Langley, B.C. was used in this experiment for all the five bulls. 3.3.7 Statistical Analysis Statistical analysis was done by General Linear Models (GLM) procedure using the SAS system (1995). Percent oocyte cleavage rate was analyzed and evaluated: (1) between bulls (2) for pooled results of different levels of sperm concentration and (3) for different levels of sperm concentrations within the bull. 3.4 RESULTS 3.4.1 Effect of Bull on cleavage rate of oocytes The cleavage rate following in vitro fertilization of bovine oocytes was analyzed after 56 - 72 h post fertilization. Significant differences were seen between bulls in cleavage of oocytes (Fig. 3.1). Out of a total 167 oocytes inseminated using semen from bull # 438, 105 cleaved. Bull # 438 proved to be the best, with a consistent cleavage rate of over 62% and was significantly different (P<0.001) from the remaining four bulls. Bull # 386 (57 out of 133 oocytes cleaved ) induced 43% cleavage and was significantly higher (P< 0.001) than bull # 257 (39 out of 142 oocytes cleaved ) (29%). Cleavage rate of bulls 391 and 398 were not different from bull 257 (table 3.1).. 41 % 70, 257 386 391 398 438 Bull # Figure 3.1 Bull effect on cleavage rate of oocyte 3.4.2 Effect of sperm concentration on cleavage rate of oocytes In the study, 20 trials were carried for each sperm concentration level of (2, 1, and 0.5 million sperm/ml). No differences were seen in cleavage rate of oocytes fertilized with 2 x 10 6 , 1 X 1 0 6 , and 0.5 X 10 6 sperm/ ml within each bull (table 3.1). Therefore the data pertaining to each concentration was combined irrespective of the bull and the pooled data from these three concentrations were tested for statistical differences and no significant differences (P>0.05) could still be established (Table 3.2). 42 Bull Sperm concentration No. of oocytes % cleaved 1 45 30.23 257 2 49 32.25 0.5 48 25.00 1 43 44.63 386 2 45 45.13 0.5 45 39.73 1 49 31.25 391 2 45 41.63 0.5 47 36.73 1 46 32.90 398 2 50 40.96 0.5 49 30.23 1 55 71.10 438 2 57 62.35 0.5 55 55.05 Table 3.1: Effect of sperm concentration within bull on cleavage rate Sperm concentration No. of oocytes % cleaved 1 million 238 42.02 2 million 246 44.47 0.5 million 244 37.35 Table 3.2: Effect of sperm concentration (Pooled data) on cleavage rate 3.4.3 Non Return Rate There was no significant (P>0.05) correlation between the non return rate in relation to AI (Table 3.3) and the first cleavage rate after IVF for each bull (r= 0.51). The fertility result after AI only varied among the bulls from 63.4% to 83.3% ( no statistical data available). The only observation seen from both AI fertility data and first cleavage following IVF was that bulls 386 and 391 which have the second and third highest cleavage rate also have the second and 43 third highest NRR results based on AI fertility data. On the contrary, bull 257 with the least cleavage rate in IVF result (29.2 %) has better NRR performance (67.3 %) than bull 438 with the highest cleavage rate in vitro result (62.8 %) and the lowest in NRR result (63.4%). Bull# % NRR # of insemination 257 67.3 278 386 73.3 176 391 72.1 172 398 83.3 132 438 63.4 147 Table 3.3. Non Return rate Results obtained from AI center Bull# % NRR % Cleavage rate 257 67.3 29.2 386 73.3 43.2 391 72.1 36.5 398 83.3 34.7 438 63.4 62.8 Table 3.4. Non Return Results and Cleavage rate 3.5 DISCUSSION It has been shown that spermatozoa obtained from different males for a particular species give different fertilization rates. This has been proven in rabbits (Brackett and Oliphant 1975), cattle (Sirard and Lambert, 1985; Iritani et al, 1986; Parrish et al, 1986; Ohgoda et al, 1988;) and other species such as sheep, pigs and goats (Cheng et al, 1986;; Mattioli et al, 1989 and Crozet et al, 1993 respectively). Likewise, Shamsuddin and Larrsone (1993) have shown that the cleavage rate of oocyte varies depending on the sperm-donor bulls in IVF. Studies conducted in sheep (Fukui et al, 1988a) and in cattle (Leibfried-Ruteledge et al, 1986; Hillery et al, 1990) showed clearly that rams and bulls selected on their merit for high fertilizing ability might be an important factor in achieving successful and consistent IVF results. First and Parrish 44 (1987) suggested that the result of IVF data together with refinement on early embryonic development might be useful to predict bull fertility. In this study, cleavage rate of zygote based on two-cell stage embryo (plate 3.1) has been examined in response to donor bull differences. Similarly, difference in sperm concentration, different levels of sperm concentration within a bull and the data obtained from field fertility test following Al ( NRR) has been compared with the first cleavage of oocytes in IVF trial to correlate bull performance. The results showed that different bulls have different abilities in influencing cleavage rate of the oocytes, but neither difference in sperm concentration nor different levels of sperm concentration for a particular bull altered cleavage rate of the oocytes. Similarly, no correlation between NRR results and first oocyte cleavage rate following IVF has been ascertained among the bulls. In the present study, in vitro matured oocytes were fertilized using frozen-thaw spermatozoa obtained from five bulls for 56-72 h., and only the cleaved embryos up to the 2-cell were recorded for each of the five bulls. The differences among bulls heavily depend on their individual attributes rather than the result of random error in semen sampling (Gordon, 1994). It has been assumed that these male variations in cleavage rate may possibly be due to differences in metabolic activity of sperm cells (Brackett and Oliphant, 1975), age (Sirard and Lambert, 1985), seminal plasma content and the ratio of seminal plasma volume to sperm number (Fukui and Ono, 1988b). Likewise, treatment of bull spermatozoa with various media and using different .heparin concentrations might give different cleavage rates as well as fertilization rates (Leibfried-Ruteledge et al, 1989). The bull variation interms of oocytes cleavage that was found in this study to support other recent reports (Lambert et al, 1984; Hanada, 1985; Aoyagi et al, 1988; Niwa and Ohgoda, 1988; Hillery et al, 1990). There are different speculations regarding the bull effect on 45 cleavage of oocytes and embryonic developments. The use of ejaculated or epididymal sperm in IVF system has shown significant differences in terms of bull variation (Goto et al, 1989). No male differences have been observed when epididymal sperm have been used in IVF; however, since seminal plasma contains decapacitation factors, variation has been seen in ejaculated sperm for IVF system. There are also notions that there is presence of some synthetic activity in oocytes that are influenced by sperm penetration, and it was believed that sperm may differ in their ability to stimulate such activities (First and Parrish, 1987). Taft et al., (1992) suggested sperm from sub fertile bulls may undergo the AR early and die prematurely. It is also suggested that the ability of sperm may differ in fertilization and subsequent embryonic development as a result of the time taken for capacitation and this time frame varies from bull to bull (Gordon, 1994). Fertilization could likely be influenced by cell cycle regulation. It has been suggested that (Parrish et al, 1992) bull sperm might influence incident of the mechanism in activating the oocyte and in triggering synthesis of compounds needed for the cell cycle. Similarly, it is believed by the same researchers that the sperm cell itself contributes something that is required for regulating the zygote cell cycle. Thus, it might be possible that different bulls contribute a different modes of action to the cell cycle in the fertilization process. Even though the effect of bull differences on cleavage rate of bovine oocytes has been demonstrated, studies carried out by others (Eyestone and First, 1989a; Brackett, et al., 1982; Miller and Hunter, 1987, Shi et al, 1990) negate this finding. It has been shown by Shi et al, (1991) analyzed the effect of bull ejaculates obtained during different seasons on cleavage rate of oocytes; yet, they were unable to establish any significant relationship between cleavage rate and bull difference. Therefore, the variations among bulls in their ability to induce oocyte cleavage might be due to other factors apart from their innate ability. These factors such as age, media, 46 capacitation agents, and other factors have to be fully investigated in the IVF system to study bull differences. Recent studies suggested that both in vivo and in vitro fertilization of bovine oocytes were unaffected by different levels of sperm concentrations (Garcia et al, 199A). In the second part of the present study, 728 oocytes were used for three levels of sperm concentration. The number of oocytes used for sperm concentration of 2, 1 and 0.5 million sperm/ ml were 246, 238 and 244 respectively. The percent cleavage rate was 44.5, 42.02 and 37.35 % for 2, 1, and 0.5 million sperm/ml concentration respectively (table 3.2). The pooled data for levels of sperm concentrations in my result clearly demonstrate that (table 3.2) even a level of 0.5 million sperm /ml gives 37.35 % cleavage rate without any statistical differences in comparison with the sperm concentration of 2 million sperm /ml which produced 44.47 % cleavage rate. In spite of differences among bulls in their inherent ability to cleave oocytes, the three sperm concentrations used in this study, 0.5, 1, and 2 million sperm / ml for all the five bulls, did not give any significant difference in terms of oocyte cleavage rate (Table 3.2). A similar result was observed by Long et al, (1993) for 0.5, 0.25, and 0.125 million sperm/ml on cleavage rate of oocytes. Bull # 438 which produced the highest cleavage rate among the five bulls was not affected by three different levels of sperm concentrations. The cleavage rate for this bull for sperm concentration of 2, 1, 0.5, million sperm / ml was 62.4, 71.1, and 55 % respectively. The cleavage rate observed for the rest of the four bulls was also not influenced by employing the aforementioned three sperm concentrations. Even though the cleavage rate for all three sperm concentrations (2, 1 and 0.5 million /ml) was not statistically significant for each bull, the cleavage rate obtained using 0.5 million sperm/ml was the lowest. In addition, the cleavage rate obtained using a sperm concentration of 2 million sperm /ml was the highest in all 47 bulls except bull #438. A concentration of 2 million sperm /ml was expected to give more acceptable cleavage rate than 0.5 and 1 million sperm/ml for all the five bulls. Nevertheless, this did not occur in this study. This study has clearly shown that a sperm concentration as low as 0.5 million sperm /ml is sufficient to influence the cleavage of oocytes in vitro. The important advantage of determining bull performance ahead of time in the AI industry is not only to predict bull fertility but also to increase the number of frozen semen straws from valuable donor bulls for economic advantage for commercial purpose (Gordon, 1994). It has been suggested that it is a common practice an ejaculated semen sample of a given sperm concentration be diluted to a certain level ( approximately 25 million sperm cells per doses) and loaded into 0.25 ml straws. It was assumed that since 50 % of sperm survive the cryopreservation process, only half of the sperm in 0.25 ml packed straw are motile. Based on this presumption, there might be a possibility to use notably lower sperm doses as low as 5 million sperm rather than 20 million sperm per straw in superior bulls. If different bulls have different performance in cleaving the oocytes in vitro (Hanada 1985), it might become a regular procedure to ascertain ideal sperm concentration to test bull's ability to fertilize an oocyte in vitro (Leibfried-Ruteledgeera/., 1989). The final part of this study has investigated the correlation between 60 - 90 d. NRR and in vitro oocyte cleavage rate for all five bulls (table 3.3 and 3.4). Despite the bull differences in oocyte cleavage rate observed in this study, no relationship could be established between in vitro oocyte cleavage rate and NRR results. According to table 3.4, it can be seen that the correlation rate between non-return rates (NRR) the in vitro oocyte cleavage rates was only 0.51 and was not significant. Even though the in vitro oocyte cleavage rates ranged from 29.2 ± 4.1 to 62.2 + 4.1 % for all the five bulls, the NRR result ranged only from 83.3 % to 63.4 % for the same 48 bulls. However, the bull which gave the maximum oocyte cleavage rate of 62.2 % in vitro (bull # 438) had the lowest NRR in vivo (63.4 %) among the five bulls. The NRR for the other three bulls were slightly correlated (without being statistically significant) with the in vitro oocyte cleavage rate. Graule et al, (1995) have suggested the existence of bull differences in vitro, yet their findings of the NRR for these bulls with IVF results were not convincing. Similarly, Ohgoda et al., (1988) supported the lack of correlation between NRR and bull fertility in vitro. In spite of a lack of proper correlation between the NRR and in vitro bull fertility (Bosquet et al, 1983), several studies have shown a direct relationship between NRR and in vitro cleavage rate for a bull (Marquant-Le-Guienne, 1989; Shamsuddin and Larsson, 1993). It has been clearly suggested that the sperm penetration of the oocytes in vitro is related to in vivo bull fertility (Hillery et al., 1990). Since the semen straws from the same collection have been used for both in vitro testing (in this study) and field trials, the possible reasons for a lack of correlation between NRR and in vitro cleavage rate could be other factors such as semen characteristics (freeze-thaw damage), media preparation, IVF system, and other factors rather than the different bulls used in this study. In summary, this study has shown that different bulls differ in their ability to produce cleaved zygotes and among the five bulls tested bull #438 and #257 gave the highest and the lowest cleavage rate, respectively. Although bulls # 438 and # 386 differed from bull # 257 in terms of their ability to cleave oocytes, bulls #391 and # 398 are not statistically different than bull # 257. In addition, the cleavage rates obtained for bull #'s 386, 391, and 398 did not differ statistically. The pooled data show that sperm concentrations of 2, 1 and 0.5 million /ml obtained for all five bulls did not affect oocyte cleavage rate. Overall, the different sperm concentrations for a particular bull (bull-sperm interaction) had no influence on oocyte 49 cleavage rate. The sperm concentration of 0.5 million /ml gave statistically similar cleavage rates as compared to 2 million sperm /ml. The 60-90 d. NRR ( table 3.4) did not correlate with the in vitro oocyte cleavage rate for all bulls. Regardless of the statistical value, the NRR for three bulls (386, 391 and 398) were slightly correlated with the in vitro cleavage rates. 3.6 CONCLUSION The spermatozoa obtained from different bulls resulted in significantly different cleavage rate of the oocytes. Since different concentration of sperm did not affect cleavage rate of the oocytes, reduced sperm concentration of 0.5 million has resulted in almost the same cleavage rate as 2 million sperm (table 3.2). Therefore, the sperm concentration of 2 million sperm / ml from superior donor bull at present used in most IVF laboratory might be reduced to 0.5 million sperm / ml to obtain similar results and successful fertilization and cleavage in vitro. There was a non-linear relationship between fertility estimate based on 60 90 d NRR and the first cleavage of the oocytes in IVF. On that account, in vivo inseminations of either superovulated or non superovulated cattle should be taken up, using reduced sperm numbers to determine if sperm per dose could be minimized in outstanding bulls, without affecting fertility.. To establish ideal correlation between the NRR and in vitro performance of bull, below average sub-fertile bulls could be used and tested before recruitment or culling by AI centers. Even though the genuine correlation between bull fertility and the outcome of IVF looked promising, it is difficult to establish if this is likely to be of value in identifying high-fertility bulls for use in commercial AI. Above all, variables involving both male and female factors, various procedure used in bull semen processing and the system of IVF must come from standard protocol to achieve success. 50 CHAPTER 4. EFFECT OF BULL ON IN VITRO EMBRYO DEVELOPMENT AND EMBRYO VIABILITY AFTER FREEZE-THAW 4.1 ABSTRACT Freeze-thawed semen of five Holstein bulls were tested. Cumulus oocyte complexes aspirated from 2-8 mm follicles (slaughterhouse ovaries) were matured in vitro in tissue culture medium (TCM 199), 5% superovulated cow serum and 0.1 A (Aromour) FSH for 21-24 h. Mature oocytes (n = 1765) with compact cumulus cells were fertilized in vitro in BO (Brackett and Oliphant; Appendix E.) media. Oocytes were washed 5 h post insemination and cocultured with cumulus cells in microdrops of TCM 199, 5% superovulated cow serum and lOpi insulin to evaluate their developmental capacity. Cleavage of oocytes to 2-cell stage and beyond was recorded at 56-72 h post insemination. After 8 days of coculture, the number of morula and blastocysts formed were recorded and the results were analyzed statistically. Embryos that developed (n = 299) to the morula and blastocyst stage (day 6 or 9) were frozen in 0.25 cc straws using 1.6 M propylene glycol and stored in liquid nitrogen. The frozen embryos were thawed in a water bath at 35 °C for 40 sec. After thawing, embryos (n = 183) were cultured for 24-28 h in cumulus cell monolayers in a culture dish containing TCM 199 supplemented with 5 % superovulated cow serum and 10 p.1 insulin under paraffin oil to assess viability. No significant differences were observed among bulls in embryo development and embryo viability after thawing (P>0.05). 5 2 4.2 INTRODUCTION Embryo development in vitro is influenced by the bull, and in particular the variations among bulls in their ability to fertilize oocytes (Leibfried-Ruteledge et ah, 1987; Kroetsch et al, 1992). It has been demonstrated that spermatozoa from different bulls influence the potential of in vitro fertilized bovine oocytes to undergo subsequent embryonic development (Eyestone and First, 1989b). These differences among bulls' ability to influence the oocyte may be broadly divided into extrinsic (Fukui et ah, 1991) such as season, maturation status of oocytes, capacitation of sperm, culturing of embryo and intrinsic such as age, sperm quality, number of ejaculates, and other factors. (Iritani et al, 1988). Prediction of male fertility based on acceptable parameters of semen evaluation is inadequate (Boyd et al., 1973) unless several parameters are considered together. Bovine embryos usually desist to develop after 8-cell stage (8-cell block) when cultured in vitro. This block has been overcome by coculturing in vitro fertilized early bovine embryo with either trophoblastic or oviductal epithelial cells (McLaughlin et al,. 1990). Similarly, blastocysts obtained after culturing with cumulus cells have resulted in the birth of viable offspring on subsequent transfer (Goto et al, 1988). Therefore, the variation between replicates are still sizable and much more research is needed to improve the rate of embryonic development in vitro. The successful production of multiple oocytes and embryos has led the research for human as well as for cattle embryo cryopreservation procedures (Blankstein, et ah, 1986). As post-thaw sperm fertility is important to the Al industry in which the fertility is directly related to bull differences (Elliot, 1978), the quality of frozen embryos must meet the good standards in embryo transfer programs which might be affected by donor (Curtis, 1991). In 1973, the first bovine calf was born following (Wilmut and Rowson, 1973 ) surgical transfer 53 of a freeze-thawed embryos. Since then, considerable progress has been made in cryopreservation techniques which allowed high survival rates for both in vivo and in vitro produced bovine embryos (Avery et al, 1992). In spite of these advances, embryo survival following cryopreservation still depends on several factors such as type of cryoprotectant, freeze-thaw procedure, time of exposure, species, stages of embryo development, and other factors. However, the effect of bull differences on freeze-thaw survival rate of embryos has not been fully established. Therefore, the objectives of this study were to assess the effect of bull differences on development of embryos in vitro and to evaluate the post-thaw survival rate of these cryopreserved embryos using propylene glycol as a cryoprotectant. 4.3 M A T E R I A L S A N D M E T H O D S 4.3.1 In V i t r o Oocyte Maturat ion: The aim of in vitro oocyte maturation (IVM) is to select those oocytes surrounded by a multilayered compact cumulus investment and homogeneous ooplasm for maturation. A typical mature oocytes has to display expanded cumulus cells, round or even ooplasm and a first polar body (although usually difficult to visualize through dense cumulus cells). In vitro oocyte maturation (IVM) incorporates collection of cumulus-oocyte-complexes (COC) and maturation of oocytes. See step wise procedure in appendix D. 4.3.1.1 Col lect ion of ovaries Ovaries were collected from cows and heifers immediately after slaughter in a local slaughter house. Healthy ovaries were placed in 0.9% (w/v) physiological saline supplemented 54 with 0.0624 g penicillin and 0.2 g streptomycin per liter saline at 30-38 °C in a thermos flask and transported to the laboratory within 3 hours from the time of collection. The ovaries were rinsed 2-3 times using physiological saline supplemented with penicillin and streptomycin before aspirating the oocytes. 4.3.1.2 Oocyte aspiration and maturation Ovaries were soaked inside a large glass beaker containing physiological saline and left in water bath (at 37 °C) for the process of aspiration. The aspiration buffer consists of phosphate buffer saline (PBS, Gibco), 6 mg/ml of bovine serum albumin (BSA) and 5 p.1 (v/v) per ml gentamycin. The pH was adjusted to 7.35 - 7.40 and filtered through a 0.2 p.m filter (Baxter Canlab, Toronto , Ont.). Oocytes were aspirated from 2-8 mm size follicles with an 18-gauge needle attached to a 10 cc syringe (Luer Lok, Rutherford, New Jersey, USA) which contained 1 cc of aspiration buffer. The aspirated oocytes were kept in a 37 °C water bath until the aspiration finished or the search under dissecting microscope started. After washing with the aspiration buffer 2-3 times, the aspirated oocytes were transferred to maturation media for selection. The maturation media was made up of TCM-199 supplemented with 0.01 A (Aromour) per ml of FSH (Cat # 101727, ICN Biomedical, Ohio, USA), 5 % (v/v) superovulated cow serum (SCS) collected from superovulated cows on day 7 of their cycle (SCS, heat inactivated), and 5p.l (v/v) per ml gentamycin. The prepared maturation media was spread in a dish, covered with oil and kept inside the incubator for almost an hour before oocytes were transferred. Oocytes with multilayered compact cumulus oocyte complex (COC's) were selected from the follicular fluid under a dissecting microscope. Multilayered (3-4 cell layers) compact cumulus oocyte complexes (COC's) were selected as a maturation stock. The selected COC's were washed 4 -5 times using the same media in a 35 mm dish 55 (Falcon 1008, Lincoln Park, NJ). Oocytes with compacted multilayer cells and uniform cytoplasmic membranes were selected and placed in a 90-ul drop of maturation medium under 1 ml of sterile light mineral oil (Sigma, USA) , which was distributed in a 4-well multidish (Nunclon Delta, Denmark) and pre equilibrated at 39 °C, 5% C 0 2 for two hours inside incubator. Finally after a 2 h., selected oocytes in 10 ul drop were added in a 4-well multidish to make a total volume of 100 ul and cultured at 39 °C, 5% C 0 2 for 22-24 h. 4.3.2 Semen Preparation and in vitro fertilization Frozen semen from the same five Holstein bulls (used in Chapter three of the previous experiment, bull # 257, 386, 391, 398, and 438) were used. Semen samples from 2 or 3 bulls were randomly allocated for rVF in each experiment. Semen straws were thawed (for 40 sec in 38 °C) and diluted to about 6-7 ml with BO- caffeine ( see appendix D.) in modified defined medium according to Brackett and Oliphants (1975) without BSA, and supplemented with 10 mM caffeine sodium benzoate. The sperm suspension was washed twice with the same amount of defined medium by centrifugation at 200 g for 5 min. each time. The supernatant was discarded leaving the pellet of sperm cells. The pellet was resuspended in about 1 ml Bo-caffeine and the sperm concentration was calculated using hemocytometer and adjusted to 20 x 106 sperm /ml (appendix F). The adjusted sperm suspension was diluted 1:1 with BO-BSA ( appendix D). Aliquots of 2 x 106/ ml were distributed in microdrops and covered with 1 ml mineral oil. The dish was incubated at 39 °C and 5% C 0 2 for 1 hour before adding the matured oocytes. The matured oocytes with expanded cumulus cells and uniform cytoplasm were removed from the maturation drops after 22-24 h., washed 2-3 times in capacitation medium (appendix D.). Only oocytes with cumulus investment were selected and picked for sperm oocyte culture. The oocytes were picked in 10 ul medium using a mouth pipette or pipetter and transferred to the 56 sperm microdrops already prepared to make the total volume 100 ul. Not more than 20 -30 oocytes were randomly added to each drop. Once the oocytes were added, the sperm and the oocytes were incubated for 5-6 h at 39 °C and 5% C 0 2 in humid air. 4.3.3 In vitro culture and Assessing Embryo Development Culture medium which consisted of TCM 199, 5% SCS or fetal calf serum (FCS), 5p.g/ml insulin and 5ur (v/v) of gentamycin were prepared 5 - 6 h. in advance at the same time as the semen sample. The medium was added to 35 mm dishes (as such or in drops) covered with oil, and left in the incubator for gassing and for later usage in washing post fertilized zygotes. Approximately 5-6 h after the fertilization, loosely adherent sperm cells were removed from the presumptive zygote by gentle rinsing 2-3 times in culture medium already prepared in 35 mm dishes. Spermatozoa that adhered to the cumulus cells were removed without removing the cumulus cells from the zygote. The zygotes were transferred into four-well culture dishes in culture medium with their intact cumulus cells. The cumulus cells were important for further embryonic development as they formed monolayers. As the cleavage and embryonic development progressed, culture medium was replaced every 48 to 72 h until blastocyst formation (day 6 or 9). Cell division and embryonic development was monitored every day until blastocyst formation. All uncleaved oocytes were removed from the media after 48-56 h. Embryonic development from 2-cell stage and beyond were observed and recorded. By the end of the experiment, embryos produced from each bull spermatozoon were recorded and expressed as developmental rate. The developmental stages from 2-cell to hatched blastocysts were shown in plate 4.1 to 4.5. 57 PLATE 4.1. IMMATURE OOCYTE IMMEDIATELY AFTER ASPIRATION AND MATURE OOCYTE WITH EXPANDED CUMULUS CELLS A) Immature oocyte immediately after aspiration (200 x). B) Mature oocyte with expanded cumulus cells obtained after 24 h of maturation. (lOOx) 58 PLATE 4.2. EARLY EMBRYONIC DEVELOPMENT. A) Four cell stage embryo approximately 3-4 days after insemination (400 x).B) Eight cell embryo approximately 4-5 days after insemination (400 x). 59 PLATE 4.4. Various stages of blastocyst A) Early blastocyst approximately 7-9 days after insemination (200 x). B) (1). Expanded and ready for hatching blastocyst approximately 7 - 9 days after insemination (2). intact empty zona (200 x). 61 PLATE 4.5. Hatching blastocyst and zona free hatched embryo A) Hatching blastocyst approximately 8-10 days after insemination (400x). B) Zona free hatched embryo (200 x). 62 PLATE 4.6. Different stages of cultured cumulus cells A) Fresh cumulus cells used for embryo culture B) Cumulus cell monolayers used for embryo culture approximately three weeks of age. 63 4.3.4 Embryo Freezing A n d Thawing Procedures for embryo freezing and thawing were done as reported by McDonald Emtech Genetics Limited. An illustrative summary of the freeze-thaw procedure is presented in Appendix G. Briefly, all the solutions were prepared in advance at working room temperature of 20 °C. Morphologically normal morula (plate 4.3) and blastocysts (plate 4.4) were used (n= 299). Embryos were suspended in Dulbecco's PBS supplemented with 10% ECS and cryopreserved with 1.6 M propylene glycol in 0.25 cc straws. Dulbecco's PBS solution (9.5 ml) and 10 % ECS (0.5 ml) were prepared. To 9 ml of the above prepared solution, 1 ml of propylene glycol and 5ul (v/v) of gentamicin/ ml was added and filtered using 0.2 um filter (Baxter Canlab, Toronto , Ont.). After spreading the filtered media into two 35 mm dishes, embryos were transferred into the first dish and left for 10 min. To accomplish washing, embryos were washed for a given period (10 min.) by moving from one point to another point inside the dish. Embryos were transferred into the second dish after 10 min and immediately loaded into 0.25 cc straws. The straws were sealed by pinching and labeled for identification to proceed to a freezing stage. The freezer machine (Bio Cool JT, FTS ® System, INC. ) was programmed to start at 0 °C 5- 10 min. and cooled to - 6 °C/min following the transfer of embryos to the machine. Forceps was immersed in liquid nitrogen well in advance for seeding purpose. Seeding straws were done by touching top of embryo column with cooled forceps. The seeding was done at -6 °C to initiate ice formation and was held at this temperature for 10 minutes. The temperature was reduced (from -6 °C) to -28 °C at rate of 0.3 °C per minute. Slow cooling was done to -36 °C at rate of 0.1 °C per minute Finally, straws were plunged into liquid nitrogen for storage. 64 Thawing was done in a 37 0 C water bath for 40 seconds. The contents of the straws were emptied into a petridish containing 4 to 5 drops of the culture media (the same composition as in vitro culture media). After washing 4-5 times with the media, the embryos were transferred to culture wells that had cumulus cell monolayers. The cumulus monolayers were saved from the previous IVF trials and stored in an incubator (plate 4.6. ). All thawed embryos were incubated in 5 % C 0 2 in air atmosphere at 38.5 °C under paraffin oil in an incubation chamber. After 24-28 h. of culture, the number of non viable and viable embryos based on zona integrity, development to expanded or hatched blastocysts respectively were recorded according to procedures reported by Suzuki et al., (1993). 4.3..5 Non return rate result The field trial conducted by the British Columbia Artificial Insemination center, Langley, B.C. was used in this experiment for all the five bulls. The in vivo fertility data was based on 60-90 day NRR to first insemination with semen from a particular bull. The number of inseminations that were done to collect the NRR for each bull varied from 132- 278 over a period of 1 to 3 years. Semen samples used for field insemination were used for in vitro studies as well. 4.3.6 Statistical Analysis For embryonic development, statistical analysis was done by General Linear Models procedure using the SAS system (1995) to determine the statistical differences among the bulls in their performance in oocyte cleavage, production of 2-cell and beyond, morula production and blastocyst formation. For the freeze-thaw survival rate, all five bulls were compared by chi-square analysis to observe if there were statistical differences among bulls in number of 65 embryos recovered after freeze-thaw, normal embryos recovered (expand or hatched), and abnormal embryos (broken zona or empty zona). A probability value of < 0.5 was considered significant. 4.4 R E S U L T S In the first part of the study, the number of oocytes cleaved were 49.8% (142/258), 60% (215/374), 57.8% (219/387), 53.2% (174/345), and 66.8 % (264/401) for bulls # 257, # 386, # 391, # 398 and # 438 respectively (Table 4.1). Among these cleaved oocytes 53.2% (73/142), 72.8% (158/215), 70.6% (162/219), 68.2% (120/174) and 60.4%(172/264) developed beyond 2 -cell stage for bull # 257, # 386, # 391, # 398 and # 438 respectively. The percentage of embryos that developed to the morula stage was 46.8% (34/73), 45.0% (67/158), 44.6% (70/162), 51.8% (58/120) and 48.8% (80/172) for bull # 257, # 386, # 391, # 398 and # 438 respectively. Finally, the percentage of embryos that developed to the blastocyst stage was 66.4% (24/34), 63.6% (42/67), 70.8% (45/70), 66.0% (38/58) and 58.2% (44/80) for bulls 257, 386, 391, 398 and 438 respectively. Out of a total of 299 embryos frozen either at the morula or blastocyst stage, 262 were recovered to study bull performance based on their ability to tolerate freeze-thaw survival rate (table 4.2). Out of 262 embryos, 183 were classified as normal (expanded or hatched) and 88 abnormal (empty or broken zona). The results of this study showed that neither embryonic development nor post thaw survival rate following cryopreservation was affected by spermatozoa from the five bulls. 66 4.4.1 Effect of bull on embryo development in Vitro No significant differences (P>0.05) were observed among bulls in embryo development. (Table 4.1). Table 4.1: Results of in vitro embryo development for five bulls Bull# % Cleaved > 2-cell stage % Morula % Blastocysts 257 47.8 53.2 46.8 66.4 386 60.6 72.8 45.0 63.6 391 57.8 70.6 44.6 70.8 398 53.2 68.2 51.8 66.0 438 66.8 60.4 48.8 58.2 Table 4.2: Non Return Rate and embryonic development Bull# % NRR % Cleaved > 2-cell stage % Morula % Blastocysts 257 67.3 47.8 53.2 46.8 66.4 386 73.3 60.6 72.8 45.0 63.6 391 72.1 57.8 70.6 44.6 70.8 398 83.3 53.2 68.2 51.8 66.0 438 63.4 66.8 60.4 48.8 58.2 4.4.2 Effect of bull on Freeze-thaw survival rate of embryos No significant differences (P> 0.05) were observed among bulls in freeze-thaw survival rate of embryos. (Table 4.3). Table 4.3: Effect of bull on freeze-thaw survival rate of embryos Bull # # recovered Normal Broken empty # # # frozen zona zona Expanded Hatched 257 31 31 21 6 4 5 1 386 56 50 35 9 6 17 3 391 56 55 32 11 12 17 5 398 85 67 51 9 7 18 2 438 71 59 39 9 11 10 2 67 4.5 DISCUSSION Cleavage rate mainly reflects the potential division of the fertilized zygote to the 2-cell stage. This cleavage alone might not be a reliable indicator of fertilization to assess the performance of bulls in vitro (Shioya, et al. 1988). Therefore, the rate of first cleavage and subsequent embryonic developmental rate of the cleaved zygote using spermatozoa from a known bull might serve as a good indicator for assessing bull performance in vitro (Barnes and Eyestone, 1990). Hillery et al, (1990) suggested the existence of bull effect on embryonic development may not be related to sperm penetration rate or the detectable normality of fertilization. Lacalandra et al., (1992) found semen of different bulls yield different fertilization rate and embryonic development. It has also been assumed in this study that not only bull performance but also freeze-thaw survival rate following cryopreservation might be an indicator for bull performance in vitro. Shi et al (1990) found differences among bulls not only in cleavage rate but also in embryonic development up to the blastocyst stage. Their results showed that individual bulls contributed differently to both fertilization and embryonic development. On the other hand, Eyestone and First (1989b) found a similarity among bulls in their ability to fertilize and cleave zygote; nevertheless, their findings supported the existence of bull variations in embryonic development, and this development was not distinct until after the first cleavage division. Similarly, Shamsuddin and Larsson (1993) have shown that bull differences existed from cleavage to the 8 cell-stage beyond which development ceased (8-cell block). There is no relationship between the rates of first cleavage and subsequent embryonic development of the cleaved oocytes with the spermatozoa from a given bull (Shamsuddin and Larsson, 1993). Both rates might act independent in their course of developmental process. 68 The variations between early cleavage and late embryonic development may be due to factors affecting fertilization and subsequent embryonic development. It is therefore necessary to take into consideration the fertilization related abnormalities among bulls that reduce the ability of embryos to develop beyond the initial cleavage stage. Oocytes with fertilization abnormalities such as polyspermy, should not have passed the 4 -cell stage. It was assumed that the first three cell divisions in bovine embryos are spontaneous (Barnes and Eye stone, 1990) because the cycles are controlled by the maternal genome carried in the oocytes. At the 4-cell stage, the embryonic genome takes control of the synthesis of macromolecules over the maternal one. Fukui et al. (1988a) have shown that a developmental block occurs at the 8-16 cell stage in ovine embryos in vitro and this block has been attributed to a male-specific effect. Similarly, bovine 8-cell stage embryos are more susceptible to suboptimal culture conditions in vitro resulting in their developmental arrest and this blockage and arrest could be related to the parental genome (Fukui et al, 1988b). It has also been suggested that embryonic death due to the male factor usually occurs only after several cleavage divisions rather than after early cleavage, ( Hillery et al., 1990). This study showed no significant differences between first cleavage and subsequent embryonic development up to blastocyst stage due to bull differences. Nevertheless, Bull #438 which gave the highest initial cleavage rate (66.8 %; Table 3.1, Chapter three) produced the lowest number of blastocyst (58.2 %.). In contrast bull # 398, even though it gave the lowest initial cleavage rate (53.2 % ), produced the highest number of morula (51.8 %). Both bull # 386 (72.8 % ) and 391 (70.6 % ) gave better early embryonic development (>2-cell); however, bull # 391 (70.8 %) produced the highest number of blastocysts development. In addition, bull # 257 with the lowest cleavage rate and early embryonic development (> 2-cell) 69 among the bulls resulted in better morula formation than bulls # 386 and 391 and better blastocyst formation than bulls # 386, 398 and 438. In my study, for example regarding bull # 438 that produced the highest initial cleavage rate but with lower embryonic development (up to the blastocyst stage) might be either due to a male specific effect or polyspermy. These factors would have led to a different results between the number of oocytes cleaved and those that developed further up to the blastocyst stage for all the bulls. However, the variations found between early and late embryonic development due to bulls was not statistically significant. This might be because all the bulls used in this study were highly fertile based on NRR. Since all sub fertile bulls at the AI center are culled before getting an opportunity to participate in the fertility trial, it would be difficult to find a wide variation between early cleavage and embryonic development due to the effect of bulls in the present study. Had sub fertile bulls been used for both this study and the field trial, a possibility for obtaining different results would have existed. Since it is known that sperm of different bulls exhibit different survival rates following cryopreservation, I decided to investigate the effect of bulls on freeze-thaw survival rate of embryos. In this study, freeze-thaw embryos produced from different bulls have no statistical significance effect on survival rate (table 4.3). Even though my results on embryo survival rate following cryopreservation were not statistically different, the normal recovered embryos were slightly different among bulls. The recovery rate of cryopreserved embryos for bulls # 398 and 438 was 77.6 % (51/67) and 74.0 % (39/59) respectively. Similarly, normal embryos (either expanded or hatched) were slightly different among bulls. Bull #438 had the second highest normal embryos among all bulls but with the least expanded and hatched embryos 27.3 % (12/39). Variations in the response of embryos to low temperatures have been demonstrated 70 both among species and within a species between developmental stages of the embryo (Polge and Willadsen 1978). Correspondingly, the type of cryoprotectant used influences embryonic development both among species and within species at different stages (Fahning and Garcia, 1992). In my study I used 1.6 M PG as a cryoprotectant. The type of cryoprotectant and the difference in molarity could have affected embryo survival rate following cryopreservation. Since method of cryopreservation and type of cryoprotectants may affect freeze-thaw survival rate of embryos, in vitro evaluation of bull performance would have been better if the cryopreservation procedure, time of exposure and cryoprotectant used had been standardized. The viability of cryopreserved embryos was assessed in this study based on their morphology in vitro. This assessment might serve as a good indicator to evaluate embryos produced by individual bulls. Since the genetic contribution made by each bull varies considerably, it would not be a reliable method to evaluate bull performance based only on embryo morphology in vitro. Instead, other parameters such as cell number, tolerance for different cryoprotectants, culture system, stages of embryonic development, and other factors might serve as a better indicator to evaluate bull performance in vitro. Finally, even though this study was unable to establish any statistical significance in embryo survival rate following cryopreservation, assumes that the results would have been better if different stages of embryos from fertile bulls as well as sub-fertile bulls had been used for this study. The correlation between the IVF results for bulls and their field fertilization rates estimated on the basis of NRR of 60-90 days after insemination has not been found in this study. The result of my study showed that bull differences could not be established by comparing embryos at both early cleavage and blastocyst stage to NRR. My findings are supported by studies of Schneider et al., (1996) and Barckett and Zuelke (1993) where they 71 found no significant relationship between in vitro embryo development and NRR. In contrast to my results Palma et al., (1996) found that bulls with low NRR had reduced ability to fertilize and produce embryos in vitro. The same result was found in France by Marquant-Le Guienne and Humblot (1992) due to bull differences in embryonic development and cleavage in relation to NRR. This correlation could be beneficial if IVF data in combination with standard semen evaluation procedures are used in the selection of young bulls with good reproductive potential since it has been found that both cleavage rate and embryonic development to the blastocyst stage are correlated with NRR (Marquant-Le Guienne and Humblot, 1992). Lack of correlation between embryonic development in vitro and NRR in my study, however is not only due to bulls but also could be due to other factors such as culture system used , capacitating agent, media supplement and many other factors. 4.6 CONCLUSION The individual bulls as a donor of spermatozoa did not affect the embryonic development in vitro. Similarly, no difference was observed in the freeze-thaw survival rate of embryos for individual bulls. The outcome of the result clearly show that all tested bulls have identical field NRR results and no correlation what so ever could be established between in NRR and IVF results. 72 CHAPTER 5. IN VITRO SYSTEM IN RELATION TO EMBRYO DEVELOPMENT 5.1 ABSTRACT To determine the bull and sperm concentration effect on bovine oocytes cleavage rate in chapter three of this thesis, cumulus oocyte complexes aspirated from 2-8 mm follicles (slaughterhouse ovaries) were matured in vitro in Hams F-10 medium supplemented with 10% estrus cow serum for 24-26 h. Approximately 150 oocytes were used per bull (n = 728). Freeze-thawed semen of five Holstein bulls was used to carry at in vitro fertilization using IVF-TALP medium (Parrish et al., 1988) along with bovine oviductal cells. Oocytes were washed 12-14 h post insemination and cultured in Ham's F-10 + 10% fetal calf serum in the presence of oviductal cells (Sivakumaran et al., 1993). Cleavage of oocytes to 2-cell stage and beyond was observed and recorded 56-72 h post insemination. In order to study bull differences on further embryonic development in chapter four of this thesis, cumulus oocyte complexes aspirated from 2-8 mm follicles (Slaughter house ovaries) were matured in vitro in tissue culture medium (TCM 199), 5% superovulated cow serum and 0.1 A FSH for 21-24 h. A total of 1765 oocytes were used for all the five bulls. Freeze-thawed semen of five Holstein bulls was used. Mature oocytes with compact cumulus cells were fertilized in vitro in BO (Brackett and Oliphant , 1975) media. Oocytes were washed 5 h post insemination and co-cultured with cumulus cells in microdrops of TCM 199, 5% superovulated cow serum and lOul insulin to evaluate their developmental capacity. Cleavage of oocytes to 2-cell stage and beyond was recorded at 56-72 h post insemination. After 8 days of coculture, the number of morulae and blastocysts formed from both methods 73 were recorded and the results were analyzed statistically. By comparing the two methods independently, I found that the BO method was superior than the TALP method in terms of embryo development in vitro (Table, 5.1 and 5.2). 5.2 INTRODUCTION AND RESULTS Oocytes matured and fertilized in vitro can develop up to blastocyst stage in different composition media under different culture conditions (Pinyopummintr and Bavister, 1991). Changes in the composition of media or in the culture environment might influence the development of the embryo to the blastocyst stage. Therefore, it is important to focus on the method of IVF used under different culture system in order to improve the media for the IVF technique. For simplicity, the method and media used are designated as TALP and BO method respectively in this study. The concentration of TALP (Parrish et al., 1988) medium used for cattle spermatozoa has been shown in appendix C. Similarly, the medium used for in vitro fertilization of rabbit spermatozoa (Brackett and Oliphant) has been shown in appendix D. In table 5.1 (TALP method), embryos produced from bull 398 reached to blastocyst stage at a higher rate than the rest of the bulls. On the other hand, bull 438 with the highest cleavage rate yielded low blastocyst development from the rest of the bulls except 391. 74 Bull# % Cleaved % Morula % Blastocysts 257 28.5 31.5 50.0 386 43.0 74.0 52.5 391 36.6 23.3 26.3 398 35.0 34.3 56.3 438 62.5 33.8 29.8 Table 5.1. Results of embryonic development for the TALP method The LSMEAN of % Cleaved, % Morula, and % Blastocysts ±4.3, ±26.9, and ±16.7 respectively In table 5.2 (BO method), the % cleaved and subsequent development was uniform for all bulls tested with the exception of bull # 438 had the highest cleavage rate but had the lowest number of blastocysts. Bull# % Cleaved % Morula % Blastocysts 257 47.8 46.8 66.4 386 60.6 45.0 63.6 391 57.8 44.6 70.8 398 53.2 51.8 66.0 438 66.8 48.8 58.2 Table 5.2. Results of embryonic development for the BO method The LSMEAN of % Cleaved , % Morula , and % Blastocysts ±8.3, ±6.5 and ± 8 respectively The results of the tables show that cleavage and subsequent embryonic development are non predictable due to complicated interrelationships in the whole in vitro system. Even though the two widely accepted sperm capacitation media have been used in this study, the following pilot study compares briefly some of the steps involved in the two IVF techniques based on culture environment. 7 5 5.3 P O S S I B L E V A R I A T I O N I N V O L V E D I N T H E T W O S Y S T E M S There are several complex processes involved in the successful production of embryos in vitro. The culture medium used in IVF system, can affect the proportion of bovine oocytes able to reach metaphase II which allows the oocyte to be fertilized by the sperm. Similarly, the adequate amount and appropriate culture media can influence subsequent embryonic development and choosing the media is crucial for IVF (Bavister et al., 1992). The culture media employed will be either simple or complex depending on the whole process of IVF from maturing oocytes to coculturing embryos. Simple media can differ in their ion concentration and energy sources; however, they are usually bicarbonate-buffered with basic physiological saline systems that have different energy sources such as pyruvate, lactate and glucose. Complex media contain the simple media, amino acids, vitamins, purines and other substances at different concentration levels to mimic cellular serum. The media are also generally supplemented with serum or albumin with trace amount of antibiotics such as penicillin, streptomycin, and gentamicin. The most commonly used complex media for IVF is a tissue culture medium called TCM 199. This medium contain Earl's salts, buffered with HEPES [N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulphonic acid)] and sodium bicarbonate supplemented with pyruvate, lactate, amino acids, vitamins, purines and other substances found in serum. On the other hand, Ham's F-10 medium consists of Krebs Ringer bicarbonate buffer system supplemented with pyruvate, lactate, amino acids, vitamins, trace elements and other substances found in serum such as thymidine and hypoxanthine. Even though many unknown substances are present in bovine cellular serum constituents, these two complex media with addition of antibodies, albumin along with serum have been widely employed for 76 IVF system. Based on this information, I used both media, TALP system and BO system, for my study and I found that the BO system with TCM 199 yields better results in IVF system. Sperm capacitation procedures are intended to stimulate the sequence of events that normally occurs in the cow's reproductive tract. The sperm from laboratory species such as rabbits and mice can be capacitated by several procedures involving different type of media. The main aim of such manipulations are the removal of surface proteins that inhibit capacitation of sperm. Spermatozoa obtained from the epididymis, freshly collected ejaculate or freeze-thawed require well established capacitation systems to optimize the success of in vitro fertilization. The TALP medium and BO medium were developed to capacitate bull and rabbit spermatozoa respectively. These two methods of capacitating system in conjunction to other parameters have been compared in this section based on their success in production of embryos in vitro. Based on current reports in literature, it has been found that mutually related factors of gonadotrophins, steroids and cellar conditions provide essential support for oocyte maturation and embryonic developments in a live cow. Furthermore, the potential influence of the oocyte on the function of somatic cells and their response to stimulation by endocrine, paracrine and autocrine factors has been indicated recently by Eppig et al., (1993). In order to mimic the cellular events of in vivo with in vitro, different types of hormones and somatic cells have been used in the IVF system. The most widely used somatic cells were cumulus cells and bovine oviductal epithelial cells as employed in my study for the BO system and TALP system respectively. The interrelationship between cells help in the passage of small molecules from one cell to another and facilitate the flow of various metabolites of low molecular weight such as 77 choline, uridin and inoitol into the oocyte membrane. The metabolic cooperation between the oocyte and co-cells serve a nutritive role during oocyte maturation and subsequent embryonic development. Staigmillar and Moor (1984) showed in sheep that the addition of supplementary cumulus cells in IVF system enabled the oocytes to undergo full maturation and normal embryonic development. The main advantage of using cumulus cells rather than oviductal cells is that cumulus cells are readily obtained as part of routine IVF activities. Oviductal secretory cells may have an important effect on facilitating both capacitation and fertilization. It has been assumed that the oviduct is not only the site of fertilization but it is also believed that it contributes to the physiological milieu in maintaining the sperm's ability to fertilize the oocyte. It has been shown by Killian and Grippo, (1992) that oviductal fluid recovered from the bovine oviduct during the estrous cycle can affect greatly the fertilizing ability of sperm. It was also found that bull sperm incubated in ampullary oviductal fluid collected close to estrus (during ovulation) has an ability to penetrate more oocytes than those incubated in other fluid samples (Xu and King, 1990). Although the use of hormone supplements in media has been controversial, most researchers currently employ them ( Saeki et al., 1990). The addition of hormones such as FSH or LH showed higher embryo yield (Brackett et al., 1989). The effect of insulin on IVF has been examined by Stubbings (1989) and found that it affects maturation, fertilization, and cleavage with higher number of cells per embryo. On the other hand Trounson et al, (1994) observed that neither gonadotrophins (FSH or LH) nor growth factors (EGF, TGF) are essential for production of high quality embryos. The success rates of either BO or TALP methods are highly variable due to the lack of a standardized protocol for in vitro maturation, fertilization and subsequent embryo culture. 78 The use of different media, hormones, energy substrates, microenvironments, inorganic and organic compounds has made the whole system more complex. The possible variations of these intricate processes are discussed briefly below. 5.3.1 In- vitro Maturation (IVM) The time interval from oocyte collection to maturation varies in different laboratories; however, a 4 h period after slaughter might create a unique microenvironment for the oocyte similar to that present near ovulation in vivo which favors adequate maturation. Ovaries were collected from a slaughter house randomly irrespective of the stage of the estrous cycle for both methods. However, in the TALP method, oviductal cells were collected from ampullary-isthmic section of oviducts from ovaries that had large dominant follicles and or lacked the presence of a corpus luteum (CL). It has been suggested that the stage of the estrous cycle might influence the cleavage rate and blastocyst formation in vitro. Boediono et ah, (1995) suggested that the number of oocytes recovered from either CL bearing or non CL bearing ovaries were similar. On the contrary, they found that cleavage rate was higher for oocytes collected from non CL bearing and blastocyst formation was higher for CL bearing ovaries. It was hypothesized that high progesterone levels during the luteal phase of the cycle might account for the better quality of embryos but not good quality cleavage due to low levels of FSH. Even though in my study the stage of the estrous cycle was not considered, the variations observed between the two methods showed that the presence of oviductal cells was not essential for higher blastocyst formation (Table 5.1). Antibiotics were added in normal saline for collecting ovaries in the BO method. The two antibiotics added were penicillin and streptomycin. The possible effects of these antibiotics on these two methods needs further investigation. So far, it is known that benzyl penicillin 79 (Penicillin G) destroys both gram-negative and positive bacteria. Since most bacteria develop resistance to antibiotics when used alone, streptomycin used along with penicillin would exert a synergistic effect in preventing the growth of microorganisms present in the media (Boyd and Hoerl, 1991). The aspiration buffer used for harvesting oocytes from ovaries in the BO system consisted of phosphate buffer saline (PBS, Gibco), BSA (6 mg/ml, Sigma) and 5ul/ml gentamycin. On the other hand, in the TALP method there was no separate aspiration media as in the BO method. The same media that was used for maturation had been used for aspiration too. Similarly, the maturation media used for the two methods differed narrowly in its composition to FSH and SCS that was included in the BO method while TALP method contained only ECS. Bovine oocytes have been successfully shown to attain maturation in vitro both in the presence (Sanbuissho and Threlfall 1990) and absence of FSH (Goto et al, 1988). The media used in TALP and BO methods were Ham's F-10 and TCM 199 respectively. The study by Lu et al, (1987) showed that TCM 199 was significantly more effective than Hams' F-10 in supporting embryo development when used in combination with the BOEC monolayer system. At the same time, Ham's F-10 was also regarded to be inferior for oocyte maturation and embryonic development (Menzo and Khatchadourian., 1991). However, an early study conducted by Rajamahendran et al, (1985) found that Ham's F-10 and steer serum was beneficial for embryo development. Conditioning the media with cow sera during in vitro maturation has given better developmental competence of embryos ( Sirard et al, 1985). The type and amount of serum added in the maturation media has been also investigated (Schellander et al, 1990) and ECS was found to give better maturation and 80 developmental rates than FCS up to the 2-cell stage. Adequate maturation and subsequent embryonic development has been obtained using 10 % rather than 15% ECS (Kim et al, 1989). Durnford et al., (1992) investigated the possibility of substituting BOEC instead of ECS for in vitro oocyte maturation and found no measurable differences between the two. Oocytes matured with either cumulus cells alone or somatic cells have resulted in better quality embryos ( Goto et al., 1988). Schneider et al., (1996) found that embryos cocultured with Buffalo rat liver cells were better in their developmental capacity to morula or blastocyst stage when compared with those cultured with BOEC alone or in cell free system media. 5.3.2 In- vitro Fertilization (IVF) The composition of organic and inorganic media used for semen preparation in both TALP and BO methods is shown in appendix C and D respectively. As essential media, either TALP or BO media has been used in semen preparation in different laboratories and has given successful fertilization rates in vitro (Suzuki et al, 1993; Brackett & Zuelke, 1993). However, the success rates obtained following the use of these two media have been found to vary considerably among laboratories. In most research laboratories high quality motile sperm are obtained by swim up separation. However, Brackett and Zuelke (1993) showed that similar fertilization results could be achieved using both swim up separated and whole semen samples. For both experiments in my study, whole semen samples were used for in vitro fertilization of oocytes. The addition of pyruvate and glucose into both BO and TALP media is assumed to be essential for the meiotic maturation in denuded but not cumulus enclosed oocytes (Susko-Parrish et al, 1992). Lactic acid, an intracellular weak acid, produced from glucose metabolism may play an important role in regulating embryo development. Supplement of D-81 glucose either on day 3 or day 4 may stimulate embryo development especially in the formation of the blastocoel (Thompson, 1996). In my study, I have used D-glucose in BO method but not in TALP method and this difference might have influenced embryonic development in the former method. In mammalian in vitro fertilization, proteins such as serum or serum albumin are commonly used in fertilization media. An optimal sperm concentration of 1 million cells/ml with or without BSA has been found to be adequate for achieving normal fertilization in vitro (Garcia et al., 1994). Even though BSA is known to exert a synergistic effect with heparin on fertilization rate, it has the disadvantage of increasing the incidence of polyspermy (Saeki, et al., 1995). Since caffeine results in an increase in sperm cAMP, the addition of caffeine along with heparin in BO method but not in TALP method have resulted in better embryo production. In this study I used 2 mg /ml heparin and 6 mg /ml of BSA in both methods. Heparin was used as a capacitating agent in both methods. Heparin concentration could have been adjusted among bulls since semen from different bulls has different tolerance for slight variation for heparin concentrations (Liebfried-Ruteledge et al., 1989), yet heparin has not been adjusted in my study for all five bull in both methods. The presence of bovine oviductal epithelial cells (BOEC) during IVF did not show an effect on cleavage rate of the embryo (Xu et al., 1992) when compared with its absence. In TALP method, I used BOEC to facilitate sperm penetration in vitro. On the other hand, cumulus cells monolayers were used in the BO method during the process of fertilization. The break through of the eight-cell developmental block in sheep embryos was first reported by Gandolfi et ah, (1986) using BOEC monolayer of culturing embryos in vitro. The development of the tissue culture technique made possible the culture of a monolayer of 82 BOEC. In vitro coculture allow scientists to study capacitation, acrosome reaction (AR) and the fertilization processes (Ellington et al, 1989). The same authors suggested that the physical contact between BOEC and sperm might help in triggering production of certain proteins that prevent premature AR and enhance gamete recognition. The studies conducted by Chen-Lu et al, (1991); Sivakumaran et al, (1991); Xu and King (1990) and King et al, (1991) found that addition of oviductal cells in suspension to TALP fertilization medium has given good quality of embryo production and subsequent development. However, Choi et al, (1991) found no beneficial effect on fertilization in the presence of BOEC in IVF medium which was similar to my result in this study. 5.3.3 In- vitro Culture (IVC) The in vitro culture technique is important for embryonic development in IVF system. A wide variety of somatic cells are able to provide a better environment for development of embryos to the blastocyst stage. In my study I separately compared the beneficial effect of BOEC and cumulus cells in their ability to produce better embryos in these two methods. The interdependency of oocyte and somatic cells for metabolism helps in maturation, fertilization and subsequent embryonic development. The function of non specific protein source in embryo culture has not been well understood. However, serum albumin has been traditionally used as a protein supplement since this protein is a major protein found in the reproductive tract. Albumin serves as means of transporting other molecules into the embryo. Since the bovine tract fluid has a variety of lipids, addition of exogenous lipids may have little impact on embryo culture. Dorland et al, (1995) have suggested cytoplasmic lipid droplets in ovine embryos associated with mitochondria may be an important source of 83 energy in vivo. Therefore, cytoplasmic lipids may act as storage fuel for embryos produced with low substrate lipid in culture media. The in-vitro matured and fertilized oocytes were cocultured with BOEC in TALP method and cumulus cells denuded from the original oocytes were used as a monolayer to culture the embryos in the case of the BO method. Embryos obtained by culture with oviduct epithelial cells (Lu et al, 1987; Fukui and Ono, 1988; Eyestone and First, 1989a; Fukui et ah, 1989 ; Nakao and Nakatsuji, 1990) can develop to morula and blastocyst stages at the rates of 33% and 26% respectively . It is believed that the presence of cumulus cells during the in vitro fertilization and subsequent embryonic development is beneficial, yet the functional role of the cells is not clear (Chian et al., 1996). The eight-cell embryo blockage was first overcome by Nakao and Nakatsuji (1990) using cumulus cells in cattle IVF system. The presence of cumulus cells including corona radiata cells during nuclear maturation did not show any obvious beneficial effect, although they may have an important function in development to the blastocyst stage in vitro (Kim et al., 1996). The coculture of bovine embryos with cumulus cell produced a higher number of embryos than using BOEC in this study. Since these two results were conducted separately for embryo production rather than comparison, it is necessary to investigate the relationship between these two methods for their embryonic production and quality in further studies. 5.4 CONCLUSION The overall success rates of in vitro embryo production will be affected by many variables involved in the system employed to achieve those successes. As a result, this study found that BO method for IVF was superior to TALP method for post insemination development of embryos derived from frozen thawed bull sperm. At any stage of the in vitro 84 process, further improvement of the methods, a basic understanding of the interactions between the gametes and the culture media is necessary for the successful IVF system. 85 CHAPTER 6. GENERAL DISCUSSION The in vitro system in the livestock industry has reduced the generation interval to select offspring from outstanding females the same way as the artificial insemination propagate the best quality of outstanding bulls (McLaren and Michie, 1956). Many reports are available describing the birth of calves with entirely in vitro system of fertilized eggs to morula or blastocyst stage in vitro (Critser et al, 1986; Hanada et al., 1986 and Lu et al, 1987). With the increased understanding of the process of sperm capacitation (Oliphant and Bracket, 1973) and with the development of in vitro fertilization techniques, several laboratory tests for assessing bull fertility have been attempted. Bousquet et al, (1983) used a zona free hamster ovum sperm penetration assay as a laboratory test for predicting bull fertility and they proposed that this method might be useful to identify subfertile bulls. Similarly, hamster oocyte penetration assays have been conducted on fresh and frozen bull semen after induction of acrosome reaction using dilauroylphosphatidyl-choline liposome (Graham and Foote, 1987a,b). Marks and Ax (1985) found a clear relationship between binding affinity of heparin to bull sperm and non return rates. In addition, Ax and coworkers (1985) reported that induction of acrosome reaction by chondroitin sulfates in vitro corresponds to non return rate of bulls. Likewise, Ax and Lenz (1986) suggested that glycosaminoglycans can serve as valuable probes in vitro to monitor cellular changes of sperm that reflect fertility of bulls. More recently in vitro fertilization tests (Marquant-Le et al, 1990) and in vivo fertilization tests using superovulated cattle have been used as methods to determine fertility of 86 bulls. It is believed that the fertilization rate may be affected by bull differences depending on the treatment of semen for IVF purposes. It was found that the presence of one chemical agent with another of its type in the media may have a synergistic effect on the fertilization rate (Saeki et al, 1995) and the incidence of polyspermy. For example, both heparin and bovine serum albumin (BSA) promote fertilization separately or act together as a synergistic effect in the process. However, addition of 0.05 pg /ml of heparin has resulted in significant correlation between IVF and NRR as well as bull differences in their ability to fertilize in vitro (Marquant-Le et al., 1990). On the other hand, the same researchers found that the optimal sperm concentration of 1 million cells/ml with and without BSA was adequate for normal fertilization in vitro. Bulls differ in their ability to penetrate and to cleave eggs in vitro and they perform in the same manner in vivo. Kroetsch and Stubbing (1992) showed that the cleavage rate of oocytes is affected by bull's gene factors and sperm concentrations. On the other hand, Eyestone and First (1989b) suggested that either bull differences or sperm concentration did not affect cleavage. Likewise, Long et al, (1993) found that sperm concentration had no effect on fertilization, however, incident of polyspermy and number of blastocysts produced increased with 0.5 million sperm/ ml. Cleavage of the embryo alone is not a reliable indicator to assess fertilization and to evaluate bull performance in vitro (see Plate 3.1 for cleavage). However, normal cleavage rate of two cell stage embryos has been used as a measure of IVF efficiency to predict fertility (Lonergan, 1992). If the first cleavage of fertilized oocytes is a good indication for the sign of fertilization, the degree of penetration of the spermatozoa to oocytes may vary from bull to bull. In the first part of the study, oocyte cleavage rate based on bull differences has been 87 seen to support other findings. The results of this study clearly showed that the performance of different bulls in their ability to cleave oocytes in vitro is due to inherent characteristic of individual bulls. Recent studies both in vivo and in vitro clearly suggest that the fertil ization of bovine oocytes was unaffected by different levels of sperm concentration. Garcia et al., (1994) have compared 20, 50 and 100 million cell by inseminating superovulated cows and after recovering embryos by non surgical methods their finding showed that 20 million sperm cells were adequate for normal embryo production with high fertility. Among the ejaculates within a bull, no differences were observed for either cleavage or embryonic development, yet bull differences were observed in blastocyst formation (Shi et al., 1991). The effect of different concentration of sperm for a particular bull did not a change in the rate of cleavage as different bulls have a different cleavage rates. Otio et al., (1993) reported that developmental capacity of embryos after insemination could be affected by factors associated with different semen straws in single lots. Similar results were obtained on in vitro oocyte cleavage rates and fertility data of the bull (Schneider et al, 1996 ). Nevertheless, different levels of sperm concentration were unable to affect oocyte cleavage rates for the same bull in this study (chapter three). Bulls which produced the highest cleavage rate (bull # 438) and least fertile bull (# 257) had different inherent nature in terms of oocyte cleavage rate. The sperm concentrations of 0.5 and 2 million sperm /ml for bull # 438 gave cleavage rate of 55.05 % and 62.35% respectively and a slightly higher cleavage rate for higher sperm concentration without any statistical significant. Consequently, the sperm concentrations of 0.5 and 2 million sperm/ml for the lowest fertile bull # 257 gave cleavage rate of 25.0% and 32.25 % respectively. It can be 88 deduced in this study that as low as 0.5 million sperm /ml can be used for low and high fertile bulls based on their cleavage rate. The rate of first cleavage and subsequent embryonic development of cleaved oocytes fertilized with spermatozoa of these bulls did not prove bull performance in this study. Bulls that have the highest potential for early stages of cleavage (bull 438, chapter three vs. chapter four) may lack the ability to develop further embryonic development to the blastocyst stage due to either early embryonic death or embryonic abnormality. Embryos produced with uniform development (bull 391) have attained their progress to the final stage of blastocysts. In addition, bull 438 gave the best cleavage but has the lowest developmental capacity, freeze-thaw sensitivity and NRR based on observations of the experimental results without statistical significant (P > 0.05). A recent studies conducted by Palma et al, (1996) showed that the results of computer-assisted analysis of spermatozoa motility and head size did not prove established correlation between in vivo and in vitro fertilizing ability. Nevertheless, the reason for lack of correlation between the in-vitro results with field insemination data is that the NRR data for all bulls in this study are very similar and sub fertile bulls would have to be tested to improve the correlation. Since the field results for all five bulls were very close, it would be hard to reach such reliable conclusions. However, observing these attributes may lead to further research to study the performance of bulls in vitro. Semen of different bulls may show different survival after cryopreservation due to permeability characteristics. It has been shown that sperm from monozygotic bulls respond similarly to different physiological stress levels in vitro. A recent studies conducted by Nishimura and Leibo (1996), have investigated differences in sperm freezing sensitivity among 89 bulls is attributable to permeability differences and whether permeability characteristics are genetically determined. In their study they found that sperm from four monozygotic bulls responds similarly to various physiological stresses. If spermatozoa from different bulls respond differently and spermatozoa from the monozygotic bulls respond similarly to cryopreservative stress, this might lead to study the possibility that embryos produced from different bulls respond differently to cryopreservation. Further investigation may be needed if embryos produced from different bulls respond differently to physiological stresses due to cryoprotectants. Since this study only analyzed embryos developed to morula and blastocyst stage following cryopreservation, it would have been ideal to study the effect of freeze-thaw survival rate of embryos in their developmental stage to assess bull differences. Takagi et al, (1993) studied survival rate of frozen-thawed bovine embryos in relation to post-thaw exposure time and they found exposure time up to 30 min. using PG was not harmful to embryos. In spite of a lack of significant relationships on embryonic development, freeze-thaw survival rate and NRR for the five bull tested, it has been observed that there are slight variations among the bulls in those three parameters that need more investigation. Budworth et al, (1988) reported that variation in computer-assisted sperm analysis parameters was limited among bulls with differences in NRR. One of the limiting factors might be that most sub-fertile bulls are culled by artificial insemination centers since keeping these bulls represent a huge expenditure for the centers. All bulls had previously sired offspring and extremely sub fertile males including those with undesirable extremes for their semen quality have not been included in this study. Therefore, it is unlikely to find sub-fertile bulls for the laboratory studies. To establish an ideal relationship in early cleavage, embryonic 90 development, freeze thaw survival rate and NRR due to bull differences, it is essential to conduct further studies of both fertile and sub fertile bulls for their performance. The fertilization process and the IVF method used to reach the goal of good quality embryo production depends on improvement of culture media as well as semen evaluation. The way the culture system in the media works is not clear at the moment. It is commonly assumed that culture systems could act as removing toxins from the media. For embryo culture media and method of IVF, the effect of culture system on embryo metabolism and appearance of possible shifts must be examined. This must be done by systematic comparison of the in vivo and in vitro metabolic features. The basic knowledge in search for early embryonic signals will allow for selection of the best embryos and success rates in improvements. On the other hand, sperm motility is an important factor in determining the fertilization rate in mammals (Mahadevan and Trouson , 1984; Tuli et al., 1992). Freezing and thawing of semen procedure might result in low motile and viable sperm due to cellular damage of spermatozoa (Hammerstedt et al., 1990). Semen collected from cattle has to be accepted for freezing when freshly obtained, and similarly has to be checked for satisfactory sperm motility and other parameters after thawing (Leidle et ah, 1993). Since 50 % or more oft he sperm may fail to survive the freeze-thawing process (Karabinus et al.., 1991), the current techniques for different diluent used on post-thaw bull semen quality has to be investigated in routine IVF procedures Therefore, assessment for cryodamage due to freeze-thaw might give a better understanding and prediction in evaluating quality of spermatozoa in vitro since bulls have different level of tolerance for freeze-thaw stress and they respond differently to cryopreservation (Elliot, 1978). These variations among bulls on oocyte cleavage rate might strengthen the other researchers findings that bull difference could be used as a tool to measure a bull performance in 91 vitro. However, further investigation is necessary to recommend if a particular male-female gamete combination can yield good cleavage rate and subsequent embryonic development. This finding and technique could be employed by AI centers to detect bulls for their performance in the field before selecting the best sires in ongoing recruitment process in the industry. 6.1 FUTURE SCOPE OF IVF The future aspect of IVF might make the field of animal production more attractive for its low cost production of large number of cattle embryos at desired stages of embryonic development for specific donor cattle. As a result, IVM, IVF, and IVC in cattle have made tremendous progress during the last 5-10 years. This technique needs to explore many areas before achieving its full potential in research and production of embryos for commercial purpose. In recent years, despite extensive research in follicular dynamics and the use of gonadotrophs hormones, successful achievement in superovulation protocols for production of embryos is still far away. Although the same statement can be said in terms of pregnancy rate for embryo transfer in cattle, the scope of improving higher pregnancy rate using IVF embryos sounds more promising than other techniques presently available. The production of good potential embryos might help in producing cattle hybrids for the tropics, twining either beef or dairy cows and in producing sexed calves for beef and dairy herds. Similarly, the IVF technique might preserve livestock breeds that are in danger of extinction all over the world. The embryo manipulation technique is one of the growing areas in the field of IVF cattle production. Some of these areas are cloning (possibility of producing copies), embryo splitting, gene transfer techniques, and microinjection to extend valuable semen to the extreme of one sperm cell per egg. 92 As the animal welfare issues increased in the public eyes, transporting farm animal from one location to other will be harder; therefore, transporting embryos will be easier than live animals. 6.2 IMPROVING IN- VITRO BOVINE EMBRYO PRODUCTION Although there has been a remarkable increase in understanding areas of the IVF process, all information is far from complete. The manipulation of the biochemical processes essential for the events still remain unknown. Nevertheless, different techniques used in IVF especially from bovine oocytes recovered from slaughterhouse material have made this system more practical not only to provide large number of embryos but also to study many areas such as egg maturation, sperm capacitation and fertilization processes in farm species. Oocyte recovery from live animals using ultrasound scanning has gained more attention in recent years. This method might be an alternate for producing embryos from valuable donors. The transvaginal ultrasound guided aspiration technique has been used for repeated recovery for collecting oocytes in a short period of time, and can be done twice weekly. Hormonal treatment of cows might lead to production of good quality of oocyte either in vivo or in vitro. The addition of prolactin might enhance the oocytes subsequent development to morula and blastocyst stage in a dose dependent manner since the level of prolactin in cows is higher in the luteal phase than the follicular phase during the estrous cycle (Yoshimura et al, 1989; Dieleman and Bevers, 1987). Other hormones such as inhibin, activin (Woodruff et al, 1990), oxytocin (Voss and Fortune, 1992), insulin (Zhang et al, 1991) and growth hormone (Sirotkin and Nitray, 1992) play an important role in maturation and embryonic development; however, much investigation is needed in these areas. Addition of growth factors to bovine embryo culture media might enhance development with successful 93 hatchability due to their autocrine and paracrine activity ( Keefer et al., 1994). It has been shown that epidermal growth factor ( EGF) stimulates the proliferation of a variety of cells in tissue culture and are potent mitogens for granulosa cells and might be beneficial for oocyte maturation (Feng et al, 1988). Although cytokines are used in the media, little information is known about their beneficial role in the IVF system. In treatment of sperm for IVF purpose, many areas have to be improved before standardizing the process. The calcium ionophore A23187, has been successfully employed for acrosome reaction and capacitation of spermatozoa. Similarly, electopermeabilization, calcium-free Tyrode's, Platelet-activating factor, liposomes, amino acids and catecholamines, chelating agents and progesterone were used as capacitating agents to induce acrosome reactions. All these chemical agents need further investigation. On the other hand, bull effect due to high and low fertility, ejaculate effect, swim-up procedure for motility of sperm, glass-wool filtration procedure for live and dead sperm, percoll density gradients for viable sperm and concentration dose dependent parameters also need further investigations. For good quality embryos production, culturing of embryos in vitro require an appropriate environment for the embryos to undergo several cleavage divisions to reach the blastocyst stage of development. During this crucial period, the embryo have to be protected from exposure to light, oxygen toxicity, and impure water sources. On the other hand, the addition of hormones, growth factors and cytokines have important regulatory effects in developmental stages of embryos, yet this area also needs more investigation. For further development of IVF technology in farm animals, much investigation is necessary for each aspect of the process in which immature oocytes can be nourished in vitro to embryonic stages that in turn can be transferred successfully into appropriate recipients for 94 normal development. Therefore, it is essential to develop basic research that explains the action of hormones and significant guidelines which involve metabolic activities through the developmental phases in optimization of defined in vitro environment to sustain these zealous processes. 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H . , (1990) : The influence of bovine oocytes type on in vitro fertilization and subsequent development in vitro. Theriogenology 33 : 355 (abst). Younis, A . I., Brackett, B . G . , Fayer-Hosken, R . A . , (1989) : Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res. 23 : 189-201. Zhang, I., Blakewood, E . G . , Denniston, R.S. and Godke, R . A . (1991): The effect of insulin on maturation and development of in vitro fertilized bovine oocytes. Theriogenology 35, 301. 109 A P P E N D I X A Appendix A . Steps involved in bovine in vitro fertilization using T A L P M E T H O D p f Steps In vitro fertilization Ovaries from slaughter housb TALP Aspiration/Slicing Oocytes matured in vitrd| (26 h at 39 c, 5% C A ) 1 / " » . . I . II I 1 TZ Cumulus cell co-culture Frozen thawed semen 10% E C S IVF TALP Fertilization 15 h Sperm TALP Hams F-10 Spermatozoa with oviductal 1 n 10% FBS H Cleavage & further development Hams F-10 20% FBS oviductal cells In vitro culture Transferable embryos 110 A P P E N D I X B Appendix B. Steps involved in bovine in vitro fertilization using BO METHOD Steps In vitro fertilization Q^esfrorn slaughter house BOrredum AspiratiorVSicing phosphate buffer saline fri (21hat39Q5%CO,) Cumiuscell co-cutture Frozen thawed semen BOIVHDIllvl Fertilization TCM 199 5 h Sperm BO Sper|TT*nmRv^th CumJus cells 5%SCS + INSUUN Cleavage & further development C C monolayer Invrtrocurtife Transferable errbryos 111 Appendix C Appendix C. Steps involved in preparation of TALPs media and its composition 1. Sperm-TALP, and Hepes-TALP (BSA with fatty acid), weigh in desired amount 2. I V F - T A L P (weigh B S A fatty acid free 20 mg /ml crystallized (Sigma) 3. Preparing primary T A L P media stock solution and stored at 4 °C 4. Preparing fresh secondary T A L P solution during the I V F trials 5. Preparing the final fresh T A L P preparation during I V F trials 6. The final T A L P is added in a desirable amount to the weighed S P - T A L P , H E P E S - T A L P , and I V F - T A L P (#1) 7. The cultured oocytes as well as the oviductal cells are washed with final media. Compositions Of T A L P Media Ingredient Units S p - T A L P Low bicarbonate- Fer t -TALP T A L P N a C l i n M 100.0 114.0 114.0 KC1 m M 3.1 3.1 3.2 N a H C 0 3 m M 25.0 2.0 25.0 N a H 2 P 0 4 m M 0.3 0.3 0.3 Lactate (sodium salt) m M 21.6 10.0 10.0 C a C l 2 m M 2.0 2.0 2.0 M g C l 2 m M 0.4 0.5 0.5 112 Ingredient Units S p - T A L P Low bicarbonate- Fer t -TALP T A L P H E P E S m M 10.0 10.0 Pyruvate m M 1.0 0.2 0.2 Glucose m M — — 5.0 B S A mg/ml 6.0 3.0 6.0 Penicillamine p.M — — 20.0 Hypotaurine u M — — 10.0 Epinephrine u M — — 1.0 Gentamycin P-g/ml 50.0 50.0 50.0 113 Appendix D Appendix D. IVF PROCEDURE FOR BO METHOD 1. Collect ovaries in normal saline with antibiotics at 30 - 32 0 C in a thermos flask-• weigh 0.9 % w/v of NaCl • add 100,0001.U of penicillin/ liter of saline solution (1602 unit = 1 mg) • add 0.2 g Streptomycin • use sterile paper towel for the process of handling ovaries N . B wash ovaries before aspiration with normal saline to avoid any blood stain 2. Aspiration buffer which consists the following chemicals: (Aspirate using 18-G needle) • Phosphate buffer 49.7 ml • B S A 6 mg/ ml • Gentamycin 50 mg/ml of buffer (5 ul / ml) • warm (heat inactivate) the media in water bath (at 38 0 C) 3. Prepare a maturation media (prepared and kept inside incubator for almost an hour). This media consists o f : • T C M 199 30 ml • Gentamycin 50 mg/ml • F S H 0.01 A / m l • SCS 5 % 4. Cover a drop of maturation medium with mineral o i l 2 or spread the maturation medium in a dish and cover with mineral oil 5. Select oocytes for maturation. Wash 6 times in maturation medium and transfer to drop/dish. If in drops, do not exceed 30 oocytes/drop of 100 ul. If in dish, 200 oocytes per 2 ml medium, covered by oil. 6. Allow the oocytes to mature for 22 h (21-24 h) in 38.5mC with 5% C 0 2 7. Prepare stock solution of medium A and medium B as follows: Medium A NaCl 4.3092 g K c l 0.1974 g C a C l 2 . H 2 0 0.2171 g N a H 2 P 0 4 . 2 H 2 0 0.0840 g M g C l 2 . 6 H 2 0 0.0697 g Phenol red (0.5% soln) 0.1 ml Distilled water to make 500 ml This medium will be YELLOW coloured. Store in 500ml bottle at 4°C. 114 Medium B • N a H C 0 3 2.5873 g • Phenol red (0.5%) 0.04 ml • Distilled water to make 200 ml Gas medium B in C 0 2 filtered through a 0.22 u filter (attached to 10 ml glass pipette) for 30 minutes. Will initially be PINK, but T U R N S Y E L L O W after gassing. Note: 1. Need not check p H or filter at this time (both A & B). 2. Medium A can be stored for 1 month or till medium B turns pink (whichever occurs earlier). 8. To prepare B O Medium for sperm capacitation and oocyte washing Step 1. a) Weigh • S O D I U M . P Y R U V A T E 0.00685 gm and • G L U C O S E 0.075 gm into a sterile 100 ml container. b) Weigh • C A F F E I N E S O D I U M B E N Z O A T E 0.02427 gm c) Weigh • B S A 0.2 gm d) Weigh • H E P A R I N 20 mg Step 2 a) A d d 38 ml of medium A and 12 ml of medium B to the Pyruvate-Glucose-container (from step 1 a). A d d 0.5ul /ml gentamicin stock. Mix thoroughly. This will be P G M E D I U M ( P Y R U V A T E - G L U C O S E ) . Colour: Y E L L O W . b) A d d 25 ml P G M E D I U M to the caffeine weighed (from step 1 b) Name this medium as B O -C A F F E I N E . M i x well and filter. Colour: PINK. c) A d d 10 ml distilled water to the heparin weighed (from step 1 d), and mix well to make 2mg/ml stock heparin. d) A d d 10 ml of P G M E D I U M to the B S A weighed (from step 1 c). Now add 26 ul of heparin stock (from step 2 c) to the P G medium + B S A mixture. Name this medium as B O - B S A . Filter but do not mix. Colour: either Y E L L O W or C O L O U R L E S S . 115 e) M i x equal volume of B O - C A F F E I N E and B O - B S A to make 6-8 ml (more if need be) [e.g.: 3 ml BO-Caffeine + 3ml of B O - B S A ] . Let us call this medium as C A P A C I T A T I O N M E D I U M . Note: Checking p H is not needed at steps 2 a, b, c, d or e. If desired, check p H and ensure it to be in the range of 7.3 to 7.5. 9. Thaw one straw of bull semen (0.25 or 0.5cc) in 30-35mC water bath for 40 sec. 10. Empty contents into 15 ml tube 11. A d d 7 ml of B O - C A F F E I N E medium to the semen 12. Centrifuge @ 1800 rpm O R Speed 4 for 5 min. (200 = 1250 R P M = setting 3 13. Discard the supernatant leaving about 1 ml. Wash the second time also with B O C A F F E I N E 14. This time, discard the supernatant completely without losing any sperm. 15. Add about 200 pi of the C A P A C I T A T I O N M E D I U M (from step 2 e) to the sperm pellet and mix well. 16. Check sperm concentration: • M i x 20 p.1 sperm suspension + 20 p.1 formal-saline • Count sperm using haemocytometer • Dilute to attain desired final concentration using capacitation medium (SEE A P P E N D I X F F O R STEPWISE P R O C E D U R E ) 17. Make sperm drop(s) of desired concentration and cover with paraffin oil 18. Observe the motility of sperm under inverted microscope. Allow one hour pre- incubation (at 38.5 °C & 5 % C 0 2 ) period if the sperm motility is good (i.e. hyperactive). If motility is poor, add oocytes immediately. 19. Pick oocytes from maturation drops and wash them gently 2-3 times using the C A P A C I T A T I O N M E D I U M (from step 2 e). 20. Transfer the oocytes into the sperm drop (@20 oocytes/drop) after 1 hour if sperm motility is good. If motility is poor, oocytes should be added immediately. 21. Incubate sperm and oocytes together for 5 hr. 116 22. Prepare culture media (5 % serum) • - T C M 199 9.5 ml • -Serum (SCS or F C S 0.5 ml • -Insulin 5 ug/ml 10 ul • -Gentamicin stock 50 ul a) May check p H (optional) and filter. b) Distribute medium in 4 well dish soon after I V F (0.5 ml medium covered by 0.5 ml oil) and leave in C 0 2 incubator for 5 h. 23. At the same time, also prepare 35 mm dishes with culture medium (as such, or in drops) covered by oil, and leave in incubator for gassing. This will be later used for washing the post fertilization oocytes. 24. At the end of 5 h incubation, wash the fertilized oocytes gently 2-3 times in the 35 mm dishes already prepared (#23). Remove sperm from cumulus cells (CC) but do not remove C C . Distribute about 30 eggs per well in the 4 well dish (from #22). Leave all the eggs in centre. C C is important, as they will form a monolayer in about 7 days. 25. Change the culture media after 48 fir. Replacement medium is the same as culture medium. Prepare medium fresh, about 2 h before the time of change and leave in incubator covered with oil, for gassing and temperature adjustment.Remove cumulus cells from embryo: Before 1st replacement of medium, make fine bore (160 p,m-size of egg) pipette. Gently pick oocytes one by one to remove cumulus cells. It is important to leave C C s in dish to form monolayer. Replace embryos on top of C C s to grow. 26. Follow the cell division and development of embryos daily. Medium change is not necessary till 7 days.Partial replacement may however be done every 48 to 72 h. 1. Day seven post oestrus cow donor which gives good embryos is preferable. Heat inactivate serum at 56 °C for 30 min. 2. Mineral oil must be gassed in C 0 2 incubator for 7 days, leaving the lid loose. 117 Appendix E Appendix E. Composition of BO media used in IVF for Embryonic development Component g/l m M N a C l 6.550 112.00 KC1 0.300 4.02 C a C l 2 . 2 H 2 0 0.330 2.25 N a H 2 P 0 4 . H 2 0 0.113 0.83 M g C l 2 . 6 H 2 0 0.106 0.52 N a H C 0 3 3.104 37.00 Glucose 2.500 13.90 Pyruvic Acid 0.110 1.25 Crystalline bovine albumin 3.000 Penicillin, sodium salt 0.031 Distilled water to 1000 ml 118 Appendix F Appendix F. Steps involved in calculating sperm concentration The formula is X (# of sperm in 5 squares) x 5 x d (dilution factor) x 10 4 = total sperm/ml. 1 2 Let us say X was 46 and X was 34. The mean of these two figures will be 46+34/2 = 40. X is 40 in this example. You have counted 40 sperm from 5 major squares. There are 25 major squares in all, in the R B C chamber. Therefore, to derive the total number of sperm in all 25 squares, multiply your count by 5. Thus, when you multiply 40 x 5, you get 200 sperm in 25 squares. Since the sperm sample was diluted 1:1 with formalinized saline, the dilution factor in this example is 2. So, you multiply the figure 200 by 2, to get 400. If there was no dilution, then the dilution factor will be 1. The multiplication factors for depth and volume corrections and to attain the concentration per ml is 104. So, you multiply the figure 400 by 104. Thus, the concentration of sperm in this example is 4 x 10 6/ml. The total volume you have is 1 ml. Therefore, the total sperm you have is 4 x 106. For I V F , we need a sperm cone, of 2 x 10 6 to 5 x 106 per ml . Since the fertilization drop size is only 100 u l , we need only 0.2 x 106 to 0.5 x 106 sperm per drop. We know that a total of 4 x 106 sperm is present in the 1 ml volume we have. We need to put in only 0.2 x 106 in each drop. 1 1 9 Calculation: 4 x 106 sperm present in 1000 ul. Therefore, 0.2 x 106 sperm will be present in 50 ul (This will be the actual volume to be added to the drop. Since this volume is unrealistically high, we need to concentrate the sperm suspension). Spin down the sperm at speed 4 for 3 minutes. Now you will see a very thin film of sperm settled down at the bottom of the tube. Discard the supernatant quickly by decanting. This will again give a 100 ul volume. It should be remembered that all the 4 x 10 6 sperm previously present in 1000 ul are now present in 100 ul volume. So it is enough to add only a tenth of the original volume, to attain the desired 0.2 x 106 sperm per drop. In the present example, therefore, you need to add only 5 p.1 of the concentrated sperm suspension to each fertilization drop, as against the 50 pi that was originally attained. N O T E : If you are using the same bull repeatedly in your experiments, you will soon attain a good idea about its post swim-up sperm concentration. Then, you may choose not to dilute the sperm to 1 ml at step 13 in Appendix D . Instead, you may check the concentration at this step itself and proceed further, thereby avoiding the second spin down. 120 Example of calculating sperm concentration for IVF (frozen vs. fresh) Given formula for frozen: Sperm con. = No of sperm counted x 5 x dilution factor x 10 4 let us say the count was 30 30 x 5 x 10 x 10 x 10 4 = 15 x 10 6 sperm per ml 1000 p.1 15 x 10 6 sperm 100 pi ? = 1.5 x 10 6 sperm for fertilization of I V F the concentration is 2 - 5 x l O 6 sperm per ml 1000 pi 5 x 10 6 sperm 100 ul ? = 0.5 x 10 6 sperm therefore, 1.5 x 1 0 6 sperm 100 p i 0.5 x 10 6 sperm ? = 33.3 ul Example for 1 x 1 0 6 sperm dilution 1000 pi 1 x 10 6 sperm 100 pi = 0.1 x 10 6 sperm then, 1 .5x10 6 sperm 100 p i 0.1 x 10 6 sperm ? = 6.7 pi 121 for 0.5 million dilution 1000 ul 0.5 x 10 6 sperm 100 p.1 ? = 0.05 x 10 6 sperm then, 1.5 x 10 6 sperm 100 p i 0.5 x 10 6 sperm ? = 3.3 ul and finally for 0.25 million sperm dilution the value will be 1.65 u,l Calculation of sperm for fresh sperm sample Given for fresh sample a) volume of ejaculate = 5 ml b) concentration = 1000 x 10 6 c) motility % = 80 calculate the following for French straws (0.25 ml dose) containing 20 x 10 6 motile sperm/dose steps i . total sperm / ejaculate = 5 x 1000 x 10 6 = 5000 x 10 6 i i . total number of motile sperm = 5000 x 0.8 x 10 6 = 4,000 x 10 6 i i i . number of straws can be made = 4,000 x l O 6 / 2 0 x l 0 6 = 200 straws iv. therefore, 200 female can be bred (number of doses) v. since volume / straw is 0.25 ml , therefore total volume will be 0.25 x 200 = 50 ml vi . amount of dilution required will then be total volume (50) - volume of ejaculate (5) =45 ml. 122 Appendix G Appendix G. Protocol for freeze-thaw survival rate of embryo and solution preparation A . Solution preparation Propylene glycol as a cryoprotectant will be used in this experiment. What are the mass and moles of propylene glycol C H 3 C H ( O H ) C H 2 O H ? Since the same of the atomic weights of all of the atoms in the molecule is expressed in molecular weight, propylene glycol has a molar mass of 76.1 g/mole. The specific gravity of propylene glycol is 1.04 g/ml. Dividing molar mass by specific gravity will give ml Mole (73.13 ml/mol). The moles of solute)/liters of solution is Molarity (M); therefore 1.0 Molar propylene glycol equal to 73.13 ml propylene glycol per 1 liter final solution. Therefore, if we need to prepare a one liter final solution of 1.6 mole propylene glycol we need 117 ml propylene glycol. B. Media preparation : In either these two methods can be prepared i . using high quality reagent 1.6 M propylene glycol in PBS + 10 % F C S (use refractometer to verify accurate mixing, with specific gravity of 1.349) • freeze in aliquot of 5 - 10 ml 123 11. propylene glycol 10% PBS 9.5 ml E C S 0.5 ml • to 9 ml of above add 1 ml of propylene glycol • add 10 ml of gentamycin • filter using 22 m filter and transfer media into two 35 ml dishes • Transfer embryo to dish one and left for 10 minutes • wash embryos during this time • Transfer to dish two after 10 minutes and load into straw • pinch open end after loading embryo with heated forceps and mark the straw C . Freezing 1. work at room temperature (20 °C) 2. start machine and program it to come to 0 °C 3. transfer straw to cool to -6 °C at a rate of 10 °C per minute 4. do a seeding at -6 0 oC . This is done by immersing pincher into liquid nitrogen and pinching the upper buffer portion (heated forceps plugged end). Total time of completion 8-12 minutes 5. observe ice formation and see progression of ice formation toward the embryo. 6. hold for 10 minutes 7. cool from -7 to -28 °C at 0.3 °C per minute 8. slow cooling from - 28 °C to - 36 °C at 0.1 °C per minute 9. transfer straw to regiform containing liquid N and then plunge straws into liquid nitrogen tank 124 D. Thawing 1. thaw straws at 35 °C water bath 2. leave in bath for 20 seconds 3. empty contents (first two columns) into 35 ml dish which contain 4 to 5 drops of culture media 4. transfer embryos from 4 - 5 drops culture media to 4-well culture dish containing cumulus cell monolayer 5. observe further embryonic development and hatching. 125 

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